JP2022540788A - Method for Protein Extraction and Downstream Processing of Euglena - Google Patents

Abstract

Aspects herein relate to methods for preparing microalgal biomass and compositions containing microalgal biomass. For example, culturing microalgae, concentrating the microalgae into microalgal biomass sludge, washing the microalgal biomass sludge, adjusting the pH of the microalgal biomass sludge, and microalgal powder. A method of preparing a microalgal flour is described herein comprising drying the microalgal biomass sludge to produce a Food products supplemented with microalgal biomass are also disclosed, including, for example, microalgal flour, protein concentrates, or protein isolates. TIFF2022540788000052.tif53170

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of US Provisional Application No. 62/868,569, filed Jun. 28, 2019, which is hereby incorporated by reference in its entirety.

SUMMARY Aspects described herein are methods of preparing a microalgal flour comprising culturing microalgae, concentrating the microalgae into a microalgal biomass sludge; A method comprising washing biomass sludge, adjusting the pH of said microalgal biomass sludge, and drying said microalgal biomass sludge to produce said microalgal flour.

In the embodiments described herein, the method of making a protein concentrate comprises culturing microalgae and reducing the culture to about 3% to about 30% solids, optionally about 10% to about 30%. % solids, optionally from about 5% to about 15% solids, adjusting the culture to a pH of about 6 to about 11, homogenizing the culture, and centrifuging the homogenate. and separating the homogenate into three layers, a pellet, a middle layer and a top layer, the middle layer being soluble protein. The middle layer is precipitated by adjusting the pH from about 3.5 to about 5.5, optionally from about 4 to about 5, optionally incubated at about 22° C. for at least 1 hour, and centrifuged to form a protein concentrate sludge (protein sludge) and a light phase called whey containing acid-soluble cellular material and non-precipitating proteins. Optionally, the protein concentrate sludge is diluted with water, optionally an equal weight of water to create a protein slurry. The pH of the protein slurry is adjusted from about 5.5 to about 8.5, optionally from about 6 to about 8. A protein slurry may be spray dried.

In the embodiments described herein, the method of producing a protein isolate comprises culturing microalgae and reducing the culture to about 3% to about 30% solids, optionally about 10% to about 10%. 30% solids, optionally about 5% to about 15% solids, adjusting the pH of the culture to about 6 to about 11, homogenizing the culture, and centrifuging the homogenate. Separating and separating the homogenate into three layers, a pellet, a middle layer and a top layer. The middle layer is precipitated by adding acid to a pH of about 3.5 to about 5.5, optionally about 4 to about 5, optionally incubated at about 22° C. for at least 1 hour, and centrifuged to produce a protein concentrate sludge. (a protein slurry) and a light phase called whey containing acid-soluble cellular material and non-precipitating proteins. The precipitated protein concentrate slurry can be washed and filtered to further increase the protein content in the protein isolate. Optionally, the protein concentrate sludge is diluted with water, optionally an equal weight of water to create a protein slurry. Optionally, the protein slurry is (a) centrifuged and (b) resuspended in water, (a) and (b) optionally being repeated one or more times. The pH of the protein sludge or slurry is adjusted from about 5.5 to about 8.5, optionally from about 6 to about 8. A protein slurry may be spray dried.

Embodiments described herein are directed to compositions comprising from about 5% to about 100% microalgal biomass.

Embodiments described herein are directed to food products comprising from about 0.1% to about 100% microalgal biomass and edible ingredients.

Solvent extracted Euglena protein concentrates are illustrated, from left to right: full fat protein concentrate, hexane defatted protein concentrate, isopropanol defatted protein concentrate, ethanol defatted protein. It is a concentrate. Figure 1 illustrates solvent extracted Euglena protein flour, from left to right: full fat protein flour, hexane defatted protein flour, isopropanol defatted protein flour, ethanol defatted protein flour. An image of a coconut citrus protein bar is shown. Images of dark chocolate, almond, and cranberry protein bars are shown. Figure 1 shows roller-stretched pasta dough containing Euglena flour, from top to bottom: 0% Euglena flour control, 10% Euglena flour, 20% Euglena flour and 30% Euglena flour. Figure 3 shows roller rolled gluten-free pasta dough with Euglena flour, from top to bottom: 0% Euglena flour control, 10% Euglena flour and 20% Euglena flour. Pasta dough containing roller-stretched Euglena flour and no egg is shown, top: 0% Euglena flour control, bottom: 20% Euglena flour. Versions 1-3 of extruded products containing protein-rich euglena flour and pea protein are shown. A shows the extruded product version 1 formulation, B is version 2 and C is version 3. Versions 4-6 of the extruded product containing protein-rich euglena flour, pea protein and masker are shown. A shows the extruded product version 4 formulation, B is version 5 and C is version 6.

Detailed Description As a civilized society, we face great challenges in the years ahead. Population growth projected over the next few decades will bring the world population to about 9.7 billion by 2050, causing severe food shortages. Malnutrition is the leading cause of death, killing about 3.5 million people annually. Global deforestation is robbing us of a significant portion of the food we currently depend on: palm oil. The resources we use today are unsustainable, and we will need the equivalent of two planets to support the projected population by 2050. Therefore, there is a great need to find sustainable alternatives. For example, food sources that can provide improved functionality, higher nutritional value, minimal waste streams, reduced water usage and reduced carbon footprint.

Microalgae are a rich source of protein, dietary fiber, essential fatty acids, vitamins and minerals. After lipid removal, the residual biomass contains even higher concentrations of protein and other nutrients. Microalgae are an excellent source of long-chain polyunsaturated fatty acids (“PUFAs”) and have been used to enrich the diet with omega-3 PUFAs. Among other things, described herein are novel techniques for extracting various components from heterotrophically grown microalgae, such as Euglena, without the use of harsh chemicals or solvents.

A particular species of algae named Euglena gracilis (hereinafter Euglena) belongs to a group of unicellular microalgae often used as candidate species for laboratory research and technical applications. Euglena has representative features typical of eukaryotic cells such as mitochondria, nuclei and lysosomes. Euglena can be further characterized by its long flagella and large red eyespots. Euglena is quite unique because it can produce its own nutrients like plants (autotrophic), but also can eat and digest external food sources like animals (heterotrophic). Euglena is a proven and versatile research model organism. Through optimizing nature's ability to use one or both aspects of nutrition, Euglena can be directed to produce target compounds by modulating key operating parameters in the production process. These important adjustments can be used to enhance the natural machinery of microorganisms to promote rapid growth and efficient conversion to valuable products with little waste generation.

There are various methods used to extract lipids, proteins and carbohydrates from cells. Some methods for extracting lipids from microalgae include organic solvent extraction (with/without Soxhlet apparatus), supercritical fluid extraction, ultrasonic or microwave assisted extraction, mechanical (i.e. press, grinding), osmotic shock or enzyme-based extraction. The extraction process needs to be optimized for microalgae due to their cellular composition. However, the process of using toxic solvents (such as hexane) defeats the purpose of extracting materials in the safest and most environmentally friendly manner. Solvent residues can also remain in the product and must be removed prior to consumer consumption. For soluble protein extraction, mechanical (bead grinding), pH, temperature or enzymatic digestion have been used to disrupt microalgal cells and subsequently purify the released proteins.

For convenience, certain terms used in the specification, examples and claims are collected here. Unless defined otherwise, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

When a range of values is provided, each intervening value between the upper and lower limits of that range, and any other stated or intervening value within the stated range, is included within this disclosure. intended to be covered. For example, if a range of 1 ml to 8 ml is stated, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml and 7 ml, and ranges of values greater than or equal to 1 ml and ranges of values less than or equal to 8 ml are also expressly stated. is intended to be disclosed in

The singular forms "a", "an" and "the" include plural references unless the context clearly indicates the contrary. Thus, for example, reference to a (a) "cell" includes a single cell as well as two or more of the same or different cells.

The word "about" when immediately preceding a numerical value means a range of plus or minus 5% of that value, unless the context of this disclosure otherwise dictates or contradicts such interpretation, e.g., " "About 50" means between 45 and 55, "about 25,000" means between 22,500 and 27,500, and so on. For example, in a list of numbers such as "about 49, about 50, about 55", "about 50" means a range that spans less than half the interval between the preceding and following values, e.g., greater than 49.5 and less than 52.5. do. Additionally, the phrases "less than about" or "greater than about" are to be understood in light of the definition of the term "about" provided herein.

The term "batch" culture refers to a culture in which cells are allowed to consume all of the medium until growth ceases, typically about two days.

By "baked good" is meant a food typically found in bakery goods prepared by using an oven and usually containing a leavening agent. Baked foods include, but are not limited to, brownies, cookies, pies, cakes and pastries.

"Bioreactor" and "fermentor" mean an enclosure or partial enclosure, such as a fermentation tank or vessel, in which cells are cultured, typically in suspension.

"Bread" means a food product containing flour, liquid, and usually a leavening agent. Bread is usually prepared by baking in an oven, but other cooking methods are acceptable. Bulking agents can be chemical or organic/biological in nature. Typically the organic leavening agent is yeast. If the leavening agent is chemical in nature (such as baking powder and/or baking soda), these foods are called "quick breads." Crackers and other cracker-like products are examples of breads that do not contain leavening agents.

The transitional phrase "comprising" is synonymous with "including," "containing," or "characterizing," including, inclusive or non-limiting, additional elements not listed. or exclude method steps. In contrast, the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of" defines the scope of a claim to the specified materials or steps of the claimed invention, "and limited to those that do not affect In embodiments or claims in which the term "comprising" is used as a transitional phrase, such embodiments refer to the term "comprising" with the term "consisting of" or "consisting essentially of". It can also be envisioned by replacing

By "cooked product" is meant food that has been heated for a period of time, eg in an oven.

"Creamy salad dressing" means a salad dressing that is a stable dispersion with high viscosity and slow pour rate. Creamy salad dressings are generally opaque.

The verbs "cultivate", "culture" and "ferment" and variations thereof refer to the growth and growth of one or more cells, typically microalgae, through the use of culture conditions. / or means intentional promotion of propagation. The intended conditions do not include microbial growth and/or propagation in nature (without direct human intervention). The term "cultured" and variations thereof refers to the growth (increase in cell size, cell contents and/or cell activity) and/or proliferation (propagation) of one or more cells through the use of the intended culture conditions. ) (increase in cell number through mitosis). A combination of both growth and propagation may be referred to under the term proliferation. One or more cells may be cells of a microorganism, such as microalgae. Examples of contemplated conditions include use of media of known composition (with known properties such as pH, ionic strength and carbon source), specified temperature, partial pressure of oxygen, carbon dioxide levels, and includes growth in

As used herein, the noun "culture" refers to a composition comprising microalgal biomass and optionally a liquid (eg, culture medium or water). For example, culture can refer to the composition of microalgal biomass, and culture can also refer to the composition of microalgal biomass and liquid medium and/or water.

"Dispersed" refers to a substantially uniform distribution of particles throughout a medium, including liquids or gases. One common form of dispersion is an emulsion made up of a mixture of two or more immiscible liquids, such as oil and water.

"Dry weight" and "dry cell weight" mean weight measured in the relative absence of water. For example, denoting a microalgal biomass as containing a specified percentage of a particular component by dry weight means that the percentage is calculated based on the weight of the biomass after substantially all of the water has been removed. means that

As used in this disclosure, the terms "emulsify," "emulsion," or derivatives thereof, refer to substances such as paramylon or food additives that are commonly It refers to being present in a food composition or food product as a phase mixture. An emulsion thus refers to a kinetically stable mixture of two normally immiscible liquids. A common example is mayonnaise, in which oil is dispersed in water. In some other foods, water is dispersed in oil.

"Edible ingredient" means any substance or composition suitable for eating. "Edible ingredients" include, but are not limited to, grains, fruits, vegetables, proteins, herbs, spices, carbohydrates and fats.

"Finished food" and "finished food ingredient" mean a food composition ready for packaging, use or consumption. For example, the "finished food" may be cooked, or the ingredients that make up the "finished food" may be mixed or otherwise integrated together. A "finished food ingredient" is typically used in combination with other ingredients to form a food product.

As used herein, the term "functional food" refers to food that has been given an additional function by adding new ingredients or more of an existing ingredient. For example, whey proteins are added to foods to give them texture, water retention capacity or nutritional support.

"food", "food composition", "food product" and "foodstuff" are intended to be ingested by humans as a source of nutrition and/or calories It means any composition expected or anticipated. Food compositions consist primarily of carbohydrates, fats, water and/or proteins and constitute substantially all of a person's daily caloric intake. A "food composition" can have a minimum weight of at least 10 times the weight of a typical tablet or capsule (a typical tablet/capsule weight range is less than or 100 mg up to 1500 mg). . A "food composition" is not enclosed or compounded in a tablet dosage form.

As used herein, the terms "gelling", "gelificate" or derivatives thereof refer to a food composition or foodstuff in gelatinous form. Gelatinous forms are created by incorporating solids and liquids into a uniform three-dimensional semi-solid structure. For example, jellies and jams, nut butters (e.g. just nuts versions), jelly-like products and fondants whose tensile strength is in the range of 500 g/cm ² to 1000 g/cm ² In some cases, gelatinous foods are considered soft gels. For example, gelatinous foods, such as those found in gummy candies, confectionery gels (i.e., cookie fillings), fruit gel bars, and fruit snacks, have a tensile strength in the range of 1000 g/cm ² to 3000 g/cm ² . Considered a hard gel.

As used herein, "foaming" refers to the ability of a material, such as a protein, to rapidly adsorb onto an air-liquid interface during foaming or foaming and form a coherent viscoelastic film through intermolecular interactions. .

As used herein, a "formulated" composition of the invention is, for example, a composition comprising (defatted) microalgae with suitable excipients, stabilizers, binders, etc. By, is meant a composition that aids in the production of stable microalgae-containing compositions suitable for oral consumption, either as a dietary or nutritional supplement or as a food additive. For example, the compositions of the invention can be formulated in solid, powder, or liquid form.

As used herein, "formulated as a food additive" means that the composition is prepared in solid form (e.g. powder, tablet, pellet) to facilitate addition to food as a food additive. etc.) or formulated in liquid form (eg, suspensions, emulsions, mixtures, etc.). For example, it may be desirable to add the composition to the food or liquid during manufacture, or it may be desirable to add the composition when the consumer prepares and/or consumes a snack or meal. . Accordingly, the specific mode of formulation will depend on the food to which the composition is added and when the composition is added.

"Current Good Manufacturing Practice" and "CGMP" refer to the regulations set forth in Title 21 C.F.R. means the conditions established by the regulatory scheme; US regulations are promulgated by the US Food and Drug Administration under the authority of the Federal Food, Drug and Cosmetic Act to regulate manufacturers, processors and packagers of food and dietary supplements for human consumption. All of the processes described herein can be performed in accordance with CGMP or equivalent regulations. In the United States, CGMP regulations for the manufacturing, packaging, or holding of human food are codified in Title 21 Part 110 of the United States Code of Federal Regulations. These provisions and the supplementary provisions referenced in these provisions are hereby incorporated by reference in their entirety for all purposes. CGMP requirements in the United States and equivalent requirements in other jurisdictions determine whether the food is not adulterated (whether the food is manufactured under conditions that make it unfit for food) or whether the food may be contaminated or otherwise Applies in determining whether food is prepared, packed, or held under unsanitary conditions that could be hazardous to health. the cleanliness and training of personnel; the maintenance and sanitary practices of buildings and facilities; the provision of adequate sanitary and housing facilities; the design, construction, maintenance and cleanliness of equipment and utensils; to ensure that all reasonable precautions are taken in the receipt, inspection, transportation, segregation, preparation, manufacture, packaging and storage of food in accordance with sound hygiene principles to prevent contamination from any source; and complying with regulations governing the storage and transportation of finished food under conditions that protect the food from physical, chemical, or unwanted microbial contamination and from deterioration of the food and its container. can include doing

"Growth" refers to changes in cell size, total cell contents, number due to cell division, and/or cell mass or weight of individual cells, including increases in cell weight due to conversion of fixed carbon sources to intracellular components. means increase.

As used herein, the term "heterotroph" or "heterotrophic environment" refers to a nutrient that is substantially completely free from exogenous sources of organic carbon such as carbohydrates, lipids, alcohols, carboxylic acids, sugar alcohols, proteins or combinations thereof. Refers to organisms such as microalgae or microorganisms, including Euglena, under conditions that allow them to obtain nutrients and energy from For example, Euglena is a heterotrophic organism that exists in an environment that is substantially devoid of light.

As used herein, the term "phototrophic" or derivatives refers to organisms, such as microorganisms, including Euglena, when the organism is present under conditions capable of performing photon capture to gain energy. point to For example, if an organism is phototrophic, it undergoes photosynthesis to produce energy.

"Homogenate" means biomass that has been physically disrupted. Homogenization is a hydromechanical process that involves breaking particles into smaller, more uniform sizes to form a dispersion that can be subjected to further processing. Homogenization is used in the processing of some food and dairy products to improve stability, shelf life, digestion and taste. "Homogenize" means to blend two or more substances into a homogenous or homogeneous mixture. In some aspects, a homogenate is produced. In other embodiments, the biomass is predominantly intact, but homogeneously distributed throughout the mixture.

As used herein, the term "hydrocolloid" refers to long-chain polymers, either carbohydrates (ie, polysaccharides) or proteins, that form viscous solutions or gels in water.

"Increased lipid yield" is defined, for example, by increasing the dry weight of cells per liter of culture, increasing the percentage of cells containing lipids, and/or It means an increase in the lipid/oil productivity of microalgal cultures that can be achieved by increasing the total amount of lipids.

"Lysate" means a solution containing the contents of lysed cells.

"Lysis" means the disruption of the plasma membrane and optionally the cell wall of a microorganism sufficient to release at least some of its intracellular contents, by mechanical or osmotic mechanisms that impair its integrity. often achieved.

By "lyse" is meant breaking down the cell membrane and optionally the cell wall of a biological organism or cell sufficient to release at least some of the intracellular contents.

"Microalgal biomass," "algal biomass," and "biomass" mean material produced by the growth and/or propagation of microalgal cells. Biomass may contain cells and/or intracellular contents as well as extracellular material. Extracellular substances include, but are not limited to, compounds secreted by cells.

A "microalgae powder" is a dry particulate composition suitable for human consumption containing cells of microalgae, eg, Euglena.

"Microalgal oil" and "algal oil" refer to any of the lipid components produced by microalgal cells, including triacylglycerols ("TAG").

By "nutraceutical" is meant a composition intended to supplement the diet by providing specific nutrients rather than bulk calories. Dietary supplements may contain any one or more of the following ingredients: vitamins, minerals, herbs, amino acids, essential fatty acids and other substances. Dietary supplements are typically tableted or encapsulated. A single tableted or encapsulated dietary supplement is typically taken at a level of 15 grams or less per day. Dietary supplements can be mixed with food compositions such as yogurt or "smoothies" to supplement the diet and are typically taken in ready-to-mix sachets (ready-to-mix) at levels of 25 grams or less per day. to-mix sachets).

"Oil" means any triacylglyceride (or triglyceride) produced by organisms including microalgae, other plants and/or animals. "Oil" as distinguished from "fat" refers to lipids that are generally liquid at normal room temperature and pressure unless otherwise specified. For example, "oil" includes soybean, rapeseed, canola, palm, palm kernel, coconut, corn, olive, sunflower, cottonseed, cuphea, peanut, camelina sativa, mustard seed, cashew, oat, Lupine, kenaf, calendula, hemp, coffee, linseed, hazelnut, euphorbia, pumpkin seed, coriander, camellia, sesame, safflower, rice, oil, cacao, copra, poppy, castor bean, pecan, jojoba, jatropha, macadamia, brazil Included are vegetable or seed oils derived from plants including, but not limited to, oils derived from nuts and avocados and combinations thereof.

"Pasteurization" means the process of heating intended to retard the growth of microorganisms in food. Typically, pasteurization is performed at elevated temperatures (but below boiling) for short periods of time. As described herein, pasteurization can not only reduce the number of undesirable microorganisms in food, but can also inactivate certain enzymes present in the food.

"Primarily intact cells" and "predominantly intact biomass" are greater than 40%, often 75%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, It means a population of cells containing more than 98% and 99% intact cells. "Intact" in this context means any cell that releases the intracellular components of the cell to the extent that the physical continuity of the cell membrane and/or cell wall surrounding the cell's intracellular components exceeds the permeability of the cell membrane in culture. It means that it is not destroyed in style either.

"Predominantly lysed" means that more than 50%, typically more than 75% to 90%, of the cell has been destroyed such that the intracellular components of the cell are no longer completely enclosed within the cell membrane means a population of cells.

"Proliferation" means a combination of both growth and propagation.

"Propagation" means an increase in cell number due to mitosis or other cell division.

As used herein, the term "shaking" refers to movement of a sample in up and down or side to side, rapid, forced, or moving and stopping motions. This can be done manually or by machine.

The term "sludge" refers to solids dispersed in a liquid to form a homogeneous composition that is not free-flowing. A composition called sludge typically contains more than 20% total solids.

The term "slurry" refers to a free-flowing composition. Compositions called slurries typically contain less than 20% total solids.

As used herein, the term "solution" refers to a homogeneous mixture of substances (solutes) dispersed throughout a liquid medium (solvent) that cannot be separated by gravity alone.

The term "substantially free" as used herein refers to a complete or nearly complete lack of light or components. For example, a composition that is "substantially free" of water is either completely devoid of water, or almost completely devoid of water, with the same effect as if it were completely devoid of water.

As used herein, the term "stability" and its derivatives refer to thermal stability, freeze-thaw stability, photostability, emulsion stability or storage stability. Thermal stability is the ability of a product or material to retain the same properties after exposure to high heat for a single set period or cycle of exposure times. Freeze-thaw stability is the ability of a product or material to retain the same properties after being frozen and subsequently thawed, and freeze-thaw can be repeated through multiple freeze-thaw cycles. Photostability is the ability of a product or material to retain the same properties after exposure to light, such as sunlight or room light, for a single set period or cycle of exposure times. Emulsion stability is the ability of a product or material to hold an emulsion and prevent it from separating over time. Additionally, the term "stabilizer" relates to materials that, when added to a product or another material, impart the stability described herein. For example, a stabilizer can be an ingredient incorporated into the final food formulation that preserves over time the structural and organoleptic properties of the food that would not be maintained in the absence of the stabilizer.

The term "solubility" as used herein generally refers to the maximum amount of a substance that can be completely dissolved in solution in a specified amount.

"Suitable for human consumption" means that the composition can be ingested by humans as a dietary intake without adverse health effects and provides significant caloric intake by uptake of digested material in the gastrointestinal tract. means that you can

"Cooked product" means a composition that has never been subjected to heat, but may contain one or more ingredients that have been previously subjected to heat.

"V/V" or "v/v" in reference to percentage by volume means the ratio of the volume of one substance in the composition to the total volume of the composition. For example, the notation of a composition comprising 5% v/v microalgal oil means that 5% of the volume of the composition is made up of microalgal oil (e.g., a composition having a volume of 100 ^mm3 is 5 mm ³ of microalgal oil), meaning that the rest of the volume of the composition (eg 95 mm ³ in this example) is made up of other ingredients.

As used herein, the term "viscosity" refers to a fluid's resistance to trying to flow and can be considered a measure of fluid friction.

"W/W" or "w/w" in relation to percentage by weight means the ratio of the weight of one substance in the composition to the weight of the composition. For example, the notation of a composition comprising 5% w/w microalgal biomass means that 5% of the weight of the composition is composed of microalgal biomass (e.g., such a composition having a weight of 100 mg is 5 mg of microalgal biomass), meaning that the remainder of the weight of the composition (eg, 95 mg in this example) is made up of other ingredients.

"W/V" or "w/v" means the ratio of the weight of one substance in a composition to the total volume of the composition. For example, the statement a composition comprising 5% w/v microalgal biomass means that 5 g of microalgal biomass is dissolved in a final volume of 100 mL of aqueous solution.

As used herein, the term "whipping" refers to the act of vigorously agitating a sample using a whisk or mixer to rapidly entrain air and cause expansion.

The term "water holding capacity" or WHC or derivatives thereof as used herein with respect to food compositions or products refers to the water content of the food itself and added water during the application of force, pressing, centrifugation or heat. Refers to the ability to retain water. WHC can also be described as a physical property, eg, the ability of a food structure to prevent water from being released from the three-dimensional structure of, for example, a gel.

Various aspects will now be described in more detail below. Such aspects may, however, be embodied in many different forms and should not be construed as limited to the forms set forth herein. Rather, these aspects are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

Methods for Preparing Microalgal Biomass Embodiments described herein are methods for preparing microalgal biomass suitable for human consumption that are rich in nutrients, including lipid and/or protein components. provide a method of combining the microalgal biomass with an edible ingredient and a food composition containing the microalgal biomass. Microalgal biomass can be prepared with high protein content and/or superior functionality, and the resulting biomass can be compared to the oil and/or protein content of the biomass present in conventional foods. It is incorporated into foods that can replace all or part of fat and/or protein, providing all essential amino acids in a highly digestible form. Microalgal oils, which may contain predominantly monounsaturated oils, provide health benefits compared to saturated, hydrogenated (trans) and polyunsaturated fats often found in conventional foods. In addition to oils and/or proteins, microalgal biomass contains several beneficial trace amounts such as algae-derived dietary fiber (both soluble and insoluble carbohydrates), phospholipids, glycoproteins, plant sterols, tocopherols, tocotrienols and selenium. It also provides nutrients. In the embodiments described herein, the microalgal biomass is in solid, powder or liquid form formulated for oral administration. In embodiments described herein, the microalgal biomass is formulated as a food additive. In embodiments described herein, microalgal biomass is processed into microalgal flour. In embodiments described herein, the protein concentrate is extracted from algal biomass. In embodiments described herein, the protein isolate is extracted from microalgal biomass.

Media and Culture Conditions for Microalgae Microalgae are cultured in liquid media to increase microalgal biomass according to the methods of the present invention. In the methods of the present invention, microalgal species are grown in a culture medium containing one or more carbon sources, one or more nitrogen sources and one or more salts in a light grown heterotrophically while maintaining a pH of about 2.0 to about 4.0 in the absence of In some species of microalgae, heterotrophic growth over extended periods of time, eg, 10-15 days or more under limited nitrogen conditions, leads to accumulation of high lipid content in the cells. Protein cell content can be varied by the carbon:nitrogen (C:N) ratio of the growth medium. A high C:N ratio favors β-glucan or carbohydrate biosynthesis. A lower C:N ratio favors protein accumulation. At production scale, carbon and nitrogen are fed separately into the tank to control the C:N ratio. The range includes C:N ratios from about 6:1 to about 80:1. Ratios of 7.2:1, 7.34:1 and 9.56:1 produce a higher percentage of protein in the biomass (from about 32% to about 49%), whereas ratios of 12:1, 20:1, 40:1 and 80:1 ratios produce lower levels of protein (about 16.7% to about 38.4%).

In embodiments, the microalgae is Euglena gracilis, Euglena sanguinea, Euglena deses, Euglena mutabilis, Euglena acus, Euglena Euglena viridis, Euglena anabaena, Euglena geniculata, Euglena oxyuris, Euglena proxima, Euglena tripteris, Euglena chlamydophora (Euglena chlamydophora), Euglena splendens, Euglena texta, Euglena intermedia, Euglena polymorpha, Euglena ehrenbergii, Euglena adhaelens ( Euglena adhaerens, Euglena clara, Euglena elongata, Euglena elastica, Euglena oblonga, Euglena pisciformis, Euglena cantabrica ), Euglena granulata, Euglena obtusa, Euglena limnophila, Euglena hemichromata, Euglena variabilis, Euglena caudata, Euglena minima, Euglena communis, Euglena communis Euglena magnifica, Euglena terricola, Euglena velata, Euglena repulsans, Euglena clavata, Euglena lata, Euglena velata Euglena tuberculata, Euglena contabrica, Euglena ascusformis, Euglena ostendensis, Chlorella autotrophica, Chlorella coloniales ( Chlorella coloniales, Chlorella lewinii, Chlorella minutissima, Chlorella pituita, Chlorella pulcheroides, Chlorella pyrenoidosa, Chlorella rotunda ), Chlorella singularis, Chlorella sorokiniana, Chlorella variabilis, Chlorella volutis, Chlorella vulgaris, Schizochytrium aggregatum , Schizochytrium limacinum, Schizochytrium minutum and combinations thereof. In certain embodiments, the microalgae is Euglena gracilis.

Aspects described herein are directed to methods of heterotrophically culturing microalgae utilizing a culture medium containing a combination of a carbon source, a nitrogen source and salts. The culture medium described utilizes all of the metabolic capabilities of microalgae, including both aerobic and anaerobic metabolism. A combination of oils, sugars, alcohols, organic and inorganic nitrogen sources results in higher conversion of input to output and faster growth of microorganisms.

In some embodiments, the growth medium may also contain exogenous nutrients and/or additives such as carbohydrates, lipids, alcohols, carboxylic acids, sugar alcohols, proteins, nitrogen, metals, vitamins, minerals or combinations thereof.

In embodiments, the carbon source is selected from oils, sugars or alcohols, carboxylic acids, potato liquor, ferulic acid and combinations thereof. In embodiments, the oil is soybean, rapeseed, canola, palm, palm kernel, coconut, corn, olive, sunflower, cottonseed, cuphea, peanut, camelina sativa, mustard seed, cashew, oat, lupine, kenaf, Calendula officinalis, hemp, coffee, linseed, hazelnut, euphorbia, pumpkin seed, coriander, camellia, sesame, safflower, rice, oil, cocoa, copra, poppy, castor bean, pecan, jojoba, jatropha, macadamia, brazil nut or avocado and Oils derived from these combinations. In one aspect, the oil is canola oil, vegetable oil, soybean oil, coconut oil, olive oil, peanut oil, fish oil, avocado oil, palm oil, flax oil, corn oil, cottonseed oil, canola oil, rapeseed oil, sunflower oil, sesame oil, grape Seed oil, safflower oil, rice bran oil, propionate and combinations thereof. Sugars may be selected from glucose, fructose, galactose, lactose, maltose, sucrose, molasses, glycerol, xylose, dextrose, honey, corn syrup and combinations thereof. Alcohols may be selected from ethanol, methanol, isopropanol and combinations thereof. In certain embodiments, the carbon source is glucose. Carboxylic acids may be selected from citric acid, citrate, fumaric acid, fumarate, malic acid, malate, pyruvate, pyruvate, succinate, succinate, acetate, acetate, lactic acid, lactate and combinations thereof.

In embodiments, the concentration of the carbon source is from about 5 g/L to about 50 g/L, from about 10 g/L to about 45 g/L, from about 15 g/L to about 40 g/L, from about 20 g/L to about 35 g/L. , about 5 g/L to about 20 g/L, about 5 g/L to about 15 g/L, about 5 g/L to about 10 g/L. In embodiments, the carbon source concentration is about 15 g/L. In embodiments, the concentration of carbon source is a concentration of about 10 g/L.

In embodiments, the nitrogen source is yeast extract, ammonium sulfate, glycine, urea, alanine, asparagine, cornsteep, liver extract, Rabo Remco, peptone, skimmed milk, soy milk, tryptone, beef extract, tricine, botanical Source peptone, pea protein, brown rice protein, soy peptone, monosodium glutamate (MSG), aspartic acid, arginine, potato broth and combinations thereof. In certain embodiments, the nitrogen source is yeast extract. In certain embodiments, the nitrogen source is ammonium sulfate. In certain embodiments, the nitrogen source is a combination of yeast extract and ammonium sulfate.

In embodiments, the concentration of the nitrogen source is from about 1 g/L to about 15 g/L, from about 1.5 g/L to about 12.5 g/L, from about 2 g/L to about 10 g/L, from about 2.5 g/L to about 8.5 g/L, about 3 g/L to about 8 g/L, about 3.5 g/L to about 7.5 g/L, about 4 g/L to about 7 g/L, about 4.5 g/L to about 6.5 g/L, or about Concentrations from 5 g/L to about 6 g/L. In embodiments, the nitrogen source concentration is about 10 g/L. In embodiments, the nitrogen source concentration is about 5 g/L. In embodiments, the nitrogen source concentration is about 2 g/L.

In embodiments, the salt is ammonium nitrate, sodium nitrate, monopotassium phosphate, magnesium sulfate, magnesium sulfate heptahydrate, calcium chloride, calcium chloride dihydrate, calcium sulfate, calcium sulfate dihydrate, calcium carbonate. , diammonium phosphate, dipotassium phosphate and combinations thereof. In certain embodiments, the salt is monopotassium phosphate, magnesium sulfate, calcium chloride.

In embodiments, the salt source concentration is about 0.01 g/L to about 5.0 g/L, about 0.1 g/L to about 4.5 g/L, about 1.0 g/L to about 4.0 g/L, about 1.5 g /L to about 3.5 g/L or about 2.0 g/L to about 3.0 g/L. In embodiments, the salt source concentration is about 0.1 g/L. In embodiments, the salt source concentration is about 1.0 g/L.

In embodiments, the culture medium further comprises a metal. Metals include iron (III) chloride, iron (III) sulfate, ferrous ammonium sulfate, ferric ammonium sulfate, manganese chloride, manganese sulfate, zinc sulfate, cobalt chloride, sodium molybdate, zinc chloride, boric acid, chloride selected from copper, copper sulfate, ammonium heptamolybdate and combinations thereof.

In embodiments, the culture medium further comprises a vitamin mixture. A vitamin mixture contains a combination of: biotin (vitamin B7), thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), pantothenic acid (vitamin B5), pyridoxine (vitamin B6), cyanocobalamin. (vitamin B12), vitamin C, vitamin D, folic acid, vitamin A, vitamin B12, vitamin E, vitamin K and combinations thereof.

Cells and/or products produced by the methods of culturing microalgae described herein are harvested at lag phase, exponential/logarithmic phase, stationary phase or death phase. In one embodiment, the cells and/or products produced are harvested or collected during the lag phase, exponential phase, stationary phase or death phase. In another embodiment, the cells and/or products produced are harvested or collected during the lag phase. In another embodiment, the cells and/or products produced are harvested or collected in log phase. In another embodiment, the cells and/or products produced are harvested or collected during stationary phase. In another embodiment, the cells and/or products produced are harvested or collected during the dying phase.

When the microalgae culture reaches stationary phase, the concentration of microorganisms in the culture reaches saturation. Saturation is determined by a number of measurements including optical density, wet cell weight, dry cell weight, cell number and/or time.

In one embodiment, the microalgae are grown to saturation measured as an optical density of about 600 nm, wet cell weight, dry cell weight or cell count. In one embodiment, the saturation as measured by optical density is from about 2 to about 10. In one embodiment, saturation as measured by wet cell weight is from about 10 g/L to about 100 g/L. In one embodiment, saturation as measured by dry cell weight is from about 2 g/L to about 50 g/L. In one embodiment, saturation as measured by cell count is from about 2.0×10 ⁶ cells/mL to about 10.0×10 ⁷ cells/mL. In one embodiment, the microorganism is grown for about 48 hours to about 350 hours, or up to about 75 days.

In general, cell culture can be classified into four culture formats: batch, fed-batch, semi-continuous and continuous culture. In batch culture, large amounts of nutrients (medium) are added to a population of cells. The cells are then grown until the input in the medium is exhausted and the desired concentration of cells is reached and/or the desired product is produced. At this point, cells are harvested and the process repeated. In fed-batch culture, medium is added at a constant rate or components are added as needed to maintain the cell population. When the maximum value or product formation is reached, most of the cells are harvested and then the remaining cells are used to start the next cycle. Fed-batch is fed with medium to bring the culture to target density when the growth fermentor is not yet full. Once full and target density is reached, continuous harvesting begins, the goal of which is to maintain a full target density culture. During semi-continuous culture, samples of a constant volume are removed at regular time intervals to make measurements and/or to collect culture components, and an equal volume of fresh medium is immediately added to the culture and immediately increases nutrient concentration and dilutes cell concentration. In continuous culture, cells are cultured in medium under conditions that allow them to be added to and removed from the medium over an extended period of time. Nutrients, growth factors and space are therefore not used up.

In one aspect, the method of heterotrophically culturing microorganisms is batch, fed-batch, semi-continuous, or continuous. In another aspect, the method of heterotrophically culturing microorganisms is batchwise. In another embodiment, the method of heterotrophically culturing microorganisms is fed-batch. In another embodiment, the method of heterotrophically culturing microorganisms is semi-continuous. In another aspect, the method of heterotrophically culturing microorganisms is continuous.

In semi-continuous and continuous cultures, fresh medium is supplied and the culture is removed from the culture vessel. Cultures can be harvested in lag phase, exponential phase or stationary phase. In one aspect, the culture is removed from the culture vessel in the lag phase, exponential phase or stationary phase. In another embodiment, the culture is removed from the culture vessel during the lag phase. In another embodiment, the culture is removed from the culture vessel during the exponential phase. In another embodiment, the culture is removed from the culture vessel at stationary phase.

For semi-continuous and continuous cultures, the culture can also be removed from the culture vessel based on time intervals. In one aspect, the culture has about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, or 192 hours, or at least 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, Removed at 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, or 192 hours. For semi-continuous and continuous cultures, a cycle is defined as tank or bioreactor turnover. Various parameters of growth are monitored and controlled within the tank or bioreactor. These include temperature, pH, dissolved oxygen level and agitation. A bioreactor or tank can be, for example, 3 L to 20,000 L. For example, bioreactors or tanks can be 3L-8L, 36L, 100L and up to 20,000L. Larger tanks are also possible, such as 100,000 L or more. In one embodiment, the tank is at least 100L, 1,000L, 10,000L, or 100,000L. In another embodiment, the tank is up to 10,000 liters, 100,000 liters, 200,000 liters, 500,000 liters or 1 million liters. Turnover is defined as emptying a container of one liquid, such as a first medium, and filling the container with a second liquid, such as a second medium. Each subsequent emptying and filling corresponds to another turnover. For example, double turnover, double turnover, or double turnover indicates that the tank has been emptied and filled twice. During continuous culture, there is substantially equal removal of culture and addition of source medium. One turnover in continuous culture is when the vessel volume is removed and refilled into the vessel. In one aspect, the method is a semi-continuous or continuous culture in a tank or bioreactor. In another embodiment, the method is a semi-continuous or continuous culture in tanks of up to 10,000 L, up to 100,000 L, up to 200,000 L, up to 500,000 L or up to 1 million L. In another embodiment, the method comprises semi-continuous or continuous culture in bioreactors up to 3 L, up to 5 L, up to 8 L, up to 10 L, up to 20 L, up to 30 L, up to 35 L, up to 36 L, up to 40 L, or up to 50 L. be. In another embodiment, the medium is turned over once, twice, three times or four times a day in the tank or bioreactor. In another embodiment, the medium is turned over up to 300 times in 75 days. In another embodiment, the medium is turned over at least 75 times, at least 150 times, at least 225 times or at least 300 times in 75 days. In another embodiment, the method is a continuous culture in a tank or bioreactor to grow the microorganisms for up to about 75 days. In another embodiment, the method is a continuous culture in a tank or bioreactor, growing the microorganisms for up to about 75 days with 300 medium turnovers. In certain embodiments, the method is a continuous culture in a tank, growing the microorganism Euglena gracilis for up to about 75 days, with 300 turnovers of the medium.

In fed-batch, semi-continuous and continuous culture, medium is added to the culture. Media can be added during the lag phase, exponential phase or stationary phase. In one aspect, medium is added to the culture during the lag phase, exponential phase or stationary phase. In another embodiment, medium is added to the culture during the induction phase. In another embodiment, medium is added to the culture during the exponential phase. In another embodiment, medium is added to the culture during stationary phase.

For fed-batch, semi-continuous and continuous cultures, medium can also be added to the culture based on time intervals. In one aspect, the medium is about 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, from the beginning of the culture or cycle of culture, or from a previous removal of the medium. 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, or 192 hours, or at least 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, Added at 164, 168, 172, 176, 180, 184, 188, or 192 hours. In another embodiment, the medium is removed from about 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours from the start of the culture or cycle of culture or from the previous removal of the medium. hours, 5 hours, 6 hours, 7 hours or 8 hours or at most 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours , 7 hours, or 8 hours. In another embodiment, medium is added at approximately the same time that medium is removed by the culture. Such additional media can be culture media, feed media, recycled culture media, spent media, replenished media and combinations thereof.

A culture medium (also known as a growth medium) is a medium that contains components required to grow or culture the cells described herein. A feed medium is a medium containing ingredients added to a culture to replenish nutrients. The feed medium is at working concentrations or concentrated levels of components to limit dilution of the culture. A feed medium is a medium containing ingredients added to a culture to replenish nutrients. The feed medium is at working concentrations or concentrated levels of components to limit dilution of the culture. Spent medium is medium that has been used for cell culture, ie, culture medium that has lower levels of growth components therein than at the start of the culture.

Spent medium is also determined by the carbohydrate content in the medium after it has been used to culture the cells. For example, about 50, 40, 30, 20, 15, 10, 8, 7, 6, 5, 4, 3, 2.5, 2, 1.5, 1, 0.5, 0.4, 0.3, 0.2, 0.1 g /L of total carbohydrates, individual carbohydrates (eg, glucose), or any combination of individual carbohydrate components (eg, glucose and maltose). Carbohydrate reduction in the spent medium can be expressed as a percentage of the starting amount of carbohydrate at the start of the culture or culture cycle. In one embodiment, the spent medium is about 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6 from the amount at the beginning of the culture or culture cycle. , containing less than 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, 0.001% total carbohydrates. In addition to carbohydrates, carboxylic acids are another carbon utilized by microorganisms. Useful carboxylic acids include citric acid, citrate, fumaric acid, fumarate, malic acid, malate, pyruvic acid, pyruvate, succinic acid, succinate, acetic acid, acetate, lactic acid and lactate. In one aspect, the spent, recycled or hybrid culture medium has less than about 20, 10, 5, 4, 3, 2, 1, 0.5, 0.4, 0.3, 0.2, or 0.1 g/L Contains carboxylic acid.

Recycled culture medium is spent medium that is used to culture cells for another passage, cycle, or to culture cells from a different culture, lot, or strain. The recycled culture medium is obtained by separating the recycled culture medium from the source culture medium, the source culture medium being in lag phase, exponential phase or stationary phase. The reused culture medium can be exclusively spent medium, or the reused culture medium can be mixed with culture medium (fresh growth medium), or one or more depleted in spent medium. Can be supplemented with more ingredients. The recycled culture medium can be obtained by separating the recycled culture medium from the source culture medium, where the source culture medium is in lag phase, exponential phase or stationary phase.

A hybrid culture medium is a culture medium that is mixed with fresh medium and recycled culture medium.

In embodiments, microalgae are grown to increase the concentration of protein in the cells. Protein cell content can be varied by the C:N ratio of the growth medium. A high C:N ratio favors β-glucan or carbohydrate biosynthesis. A lower C:N ratio favors protein accumulation. At production scale, carbon and nitrogen are fed separately into the tank to control the C:N ratio. C:N ranges from 6:1 to about 80:1. C:N ratios of about 7.2:1, about 7.34:1 and about 9.56:1 produce a higher percentage of protein in the biomass (about 32% to about 49%), whereas about 12:1, Higher C:N ratios of about 20:1, about 40:1 and about 80:1 produce lower levels of protein (about 16.7% to about 38.4%). During the lag phase of cell growth, carbon removal prior to culture can increase protein. pH plays a role in protein accumulation in Euglena. At acidic pH (about 1 to about 5), the protein concentration in Euglena is high. At neural pH (about 5 to about 8), protein concentration is at intermediate levels, except at pH 7, where there is an increase in protein concentration. Protein concentrations are lowest at basic pH (about 9 to about 14).

High-protein biomass derived from algae is an advantageous material for inclusion in food products. The method of the present invention is about 20% to about 90%; A biomass having an amount of protein as measured by % of dry cell weight selected from the group consisting of about 55%, about 60% to about 90% and about 60% to about 80% can also be provided.

In one embodiment, the biomass has a chlorophyll content of less than 200 ppm. Heterotrophic growth results in relatively low chlorophyll content (compared to phototrophic systems such as open ponds or closed photobioreactor systems). A reduced chlorophyll content generally improves the organoleptic properties of microalgae, thus allowing more algal biomass to be incorporated into foods. The reduced chlorophyll content found in heterotrophically grown microalgae also reduces the green color in the biomass compared to phototrophically grown microalgae. Thus, reduced chlorophyll content avoids the often undesirable green coloration associated with foods containing phototrophically grown microalgae and allows incorporation or increased incorporation of algal biomass into foods. do.

Harvesting or Concentrating Microalgae After Fermentation Microalgal cultures produced according to the methods described herein yield microalgal biomass in the fermentation broth/medium. Biomass is concentrated or harvested from the fermentation medium to prepare the biomass for use as a food composition. At the time the microalgal biomass is harvested from the fermentation medium, the biomass comprises predominantly intact cells suspended in the aqueous culture medium.

A dehydration step is performed to concentrate the biomass. Dewatering refers to separating biomass from a fermentation broth or other liquid medium to produce sludge. In aspects described herein, during dehydration, the culture medium may be removed from the biomass by a method selected from the group consisting of decantation, centrifugation, filtration, or combinations thereof. The dehydration process concentrates the biomass to about 1% to about 15% or about 10% to about 30% solids. In one embodiment, microbial harvesting is completed by sedimenting the cells. On a larger scale, the microorganisms are allowed to settle to the bottom of the tank in order to separate the cells from the source medium. The microorganisms are then removed from the bottom of the tank, leaving the remaining spent medium in the tank. The tank can then be replenished with fresh growth medium, recycled culture medium, or a mixture thereof.

Collection of microorganisms can also be established by mechanical methods such as centrifugation. Centrifugation involves the use of centrifugal force to separate mixtures. During centrifugation, the higher density components of the mixture move away from the axis of the centrifuge, while the lower density components of the mixture move toward the axis. By increasing the effective gravitational force (i.e. by increasing the centrifugation speed, or rotor arm length), higher density materials such as solids will separate from lower density materials such as liquids, thus increasing the density separate according to The centrifugation process can be carried out either in batch mode using a floor centrifuge or in continuous mode with a continuous centrifuge. Centrifugation of biomass and broth or other aqueous solutions forms a concentrated sludge containing mainly microalgal cells.

Membrane filtration can also be used for dehydration. One example of membrane filtration suitable for the present invention is tangential flow filtration (TFF), also known as cross-flow filtration. Tangential flow filtration is a separation technique that uses membrane systems and flow forces to separate solids from liquids based on the selective exclusion of particles larger than the nominal pore size of a filter element.

Dehydration is performed using mechanical pressure applied directly to the biomass to separate the liquid fermentation broth from the microalgal biomass sufficient to dehydrate the biomass but not cause significant lysis of the cells. can also be done. Mechanical pressure to dewater the microalgal biomass can be applied using, for example, a belt filter press.

In the embodiments described herein, the dehydrated microalgal biomass consists primarily of intact cells. In embodiments, the content of intact cells in the dehydrated microalgal biomass is from about 25% to about 99%, from about 30% to about 99%, from about 35% to about 99%, from about 40% to About 99%, about 45% to about 99%, about 50% to about 99%, about 55% to about 99%, about 60% to about 99%, about 65% to about 99%, about 70% to about 99% %, about 75% to about 99%, about 80% to about 99%, about 85% to about 99%, about 90% to about 99%, or about 95% to about 99%.

Harvesting of microorganisms in culture can be done in whole or in part. In one embodiment, the harvested microorganisms in the culture are about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20% of the total source culture , about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100%.

After concentration, the dehydrated microalgal biomass can be further processed to produce a microalgal flour, protein concentrate or protein isolate, as described herein. Alternatively, the microalgal biomass can be temporarily refrigerated or frozen for later use or processing.

Microalgal biomass can be utilized in the preparation of the food products described herein. For example, any microalgal biomass described herein can be combined with other ingredients described herein to form a food product. For example, wet microalgal biomass (e.g., that has not been subjected to drying as described herein), alone or in combination with other ingredients, as a food product as described herein, or It can be used to prepare the food products described herein.

Method of Preparing Microalgal Flour In embodiments described herein, the method for preparing microalgal flour comprises culturing microalgae and concentrating said microalgae into microalgal biomass sludge. washing the microalgal biomass sludge; adjusting the pH of the microalgal biomass sludge; and drying the microalgal biomass sludge to produce the microalgal flour. . In some embodiments, the microalgae are Euglena.

In embodiments described herein, a method for preparing microalgal flour comprises culturing microalgae, concentrating said microalgae into a microalgal biomass sludge, and washing the sludge. In embodiments, the microalgal biomass sludge is washed about 1 to about 3 times, optionally about 2 times. In embodiments, the microalgal biomass sludge is washed from about 1 to about 4, optionally about 2.5 times the volume of the sludge.

In embodiments described herein, the method for preparing microalgal flour further comprises adjusting the pH of the microalgal biomass sludge. In embodiments, the pH of the microalgal biomass sludge is adjusted to about 9 to about 11, about 7 to about 10, or about 6.5 to about 7.5. In embodiments, the pH of the microalgal biomass sludge is adjusted to about 7.

In embodiments described herein, microalgal biomass sludge is dried to produce a protein-rich flour. Microalgal biomass sludge consists mainly of intact cells. In embodiments, the content of intact cells in the microalgal biomass sludge is from about 25% to about 99%, from about 30% to about 99%, from about 35% to about 99%, from about 40% to about 99%. , about 45% to about 99%, about 50% to about 99%, about 55% to about 99%, about 60% to about 99%, about 65% to about 99%, about 70% to about 99%, about 75% to about 99%, about 80% to about 99%, about 85% to about 99%, about 90% to about 99%, or about 95% to about 99%.

In embodiments, concentrated microalgal biomass sludge is drum dried into flake form to produce algal flakes. In another embodiment, the concentrated microalgal biomass sludge is spray dried or flash dried to form a powder containing predominantly intact cells to produce a protein-rich flour. Drum drying is a process by which wet materials can be dried in the form of very thin films. This drying process typically utilizes a cylinder or drum mounted on a horizontal shaft and capable of rotating at various controlled speeds. The drum is internally heated by steam, which condenses on the inner surface, thereby creating a drying effect through the transfer of heat from inside the drum through the metal wall to a thin layer of material on the outer surface. The internal drum pressure typically ranges from about 200 kPa to about 500 kPa and the external drum temperature reaches about 120°C to about 155°C. Once dried, the microalgae flour, flakes or powder is removed from the surface of the drum using a blade. This process is typically used to make a variety of food products such as, but not limited to, soups, instant mashed potatoes, precooked cereals and low grade milk powders and other thin powdered ingredients. .

In embodiments, the microalgal flour, flakes or powder is 15% or less, 10% or less, 5% or less, 2% to 6% or 3% to 5% by weight after drying. is the moisture content of

In the embodiments described herein, the amount of protein in the microalgal flour, flakes or powder by dry weight is from about 25% to about 55%, from about 30% to about 50%, or from about 35% to about Selected from the group consisting of 45%.

Methods of Preparing Protein Concentrates and Protein Isolates In embodiments described herein, a method of making a protein concentrate comprises culturing microalgae and converting the culture to form biomass. from about 1% to about 30% solids, optionally from about 10% to about 30% solids, optionally from about 5% to about 15% solids; homogenizing the biomass; centrifuging the homogenate; separating the homogenate into three or more layers, such as a pellet, an aqueous middle layer and a top lipid-enriched layer. and the aqueous intermediate layer contains soluble proteins. Pellets may contain more than one layer that is physically separated from aqueous and insoluble components. Soluble proteins are optionally precipitated by adding acid to adjust the pH to about 3.5 to about 5.5, optionally about 4 to about 5, optionally incubated at about 22° C. for at least 1 hour, and centrifuged. This gives a heavy phase made from protein concentrate sludge (protein sludge) and a light phase called whey containing acid soluble cellular material and non-precipitated proteins. Optionally, the homogenate can also be centrifuged, yielding soluble protein directly as a protein concentrate sludge as a component of the pellet. Optionally, the pelleted protein concentrate sludge is physically separated from other components of the pellets. Optionally, the protein concentrate sludge is diluted with water, optionally an equal weight of water, to make the protein concentrate slurry. The pH of the protein concentrate sludge or protein concentrate slurry is adjusted to about 5.5 to about 8.5, optionally about 6 to about 8. The protein concentrate slurry can be spray dried. In some embodiments, the microalgae are Euglena.

In embodiments, the protein concentrate is about 40% to about 85%, about 45% to about 80%, about 50% to about 75%, about 50%, about 70%, about 55% to It has a protein concentration of about 65% or about 70%.

In embodiments, the pellet has a beta-glucan concentration of about 80% to about 95%, optionally greater than 95%.

In embodiments, the protein concentrate is defatted using an organic solvent, thereby increasing the protein content to greater than 80%. In embodiments, the organic solvent is acetone, benzyl alcohol, 1,3-butylene glycol, carbon dioxide, castor oil, citrate esters of mono- and diglycerides, ethyl acetate, ethyl alcohol (ethanol), ethyl denatured with methanol. Alcohol, glycerol (glycerin), glyceryl diacetate, glyceryl triacetate (triacetin), glyceryl tributyrate (tributyrin), hexane, isopropyl alcohol (isopropanol), methyl alcohol (methanol), methyl ethyl ketone (2-butanone), methylene chloride (dichloromethane) ), mono- and diglycerides, citric acid monoglycerides, 1,2-propylene glycol (1,2-propanediol), propylene glycol mono- and diesters of lipogenic fatty acids, triethyl citrate and combinations thereof .

In the embodiments described herein, the method of producing a protein isolate comprises culturing microalgae and reducing the culture to about 1% to about 30% solids, optionally about 10% to about 10%. 30% solids, optionally about 5% to about 15% solids, adjusting the pH of the culture to about 6 to about 11, homogenizing the culture, and centrifuging the homogenate. Separating and separating the homogenate into three layers, a pellet, a middle layer and a top layer. The middle layer is precipitated by adding acid to a pH of about 3.5 to about 5.5, optionally about 4 to about 5, optionally incubated at about 22° C. for at least 1 hour, and centrifuged to produce a protein concentrate sludge. (protein sludge) and a light phase called whey containing acid-soluble cellular material and non-precipitated proteins. The precipitated protein concentrate sludge can be washed and filtered to further increase the protein content in the protein isolate. Optionally, the protein concentrate sludge is diluted with water, optionally an equal weight of water to create a protein slurry. Optionally, the protein slurry is (a) centrifuged and (b) resuspended in water, (a) and (b) optionally being repeated one or more times. The pH of the protein sludge or slurry is adjusted from about 5.5 to about 8.5, optionally from about 6 to about 8. A protein slurry may be spray dried. In some embodiments, the microalgae are Euglena.

In embodiments, the protein isolate is prepared from protein concentrate sludge or powder. Optionally, the protein isolate is prepared by extracting lipids from the protein concentrate using a solvent. Optionally, the solvent is acetone, benzyl alcohol, 1,3-butylene glycol, carbon dioxide, castor oil, ethyl acetate, ethyl alcohol, glycerol, glyceryl diacetate, glyceryl tributyrate, hexane, isopropyl alcohol, methyl alcohol, methyl ethyl ketone, Selected from the group consisting of methylene chloride, 2-nitropropane, 1,2-propylene glycol, propylene glycol mono- and di-esters, triethyl citrate and combinations thereof. Optionally the solvent used is hexane, ethanol or isopropanol. Optionally, the solvent used is isopropanol. Optionally, solvent is removed from the protein isolate.

In embodiments, the protein isolate has a protein concentration of at least 40%, at least 80%, at least 85%, at least 90%, or at least 95% by dry weight.

In embodiments, the pellet has a beta-glucan concentration of about 80% to about 95%, optionally greater than 95%.

In aspects described herein, the method for preparing the protein concentrate or protein isolate comprises culturing the microalgae described herein.

In embodiments described herein, bringing the culture to a certain percent solids content can include, for example, reconstituting previously cultured microalgal biomass (e.g., Euglena). In some embodiments, reconstituting the previously cultured microalgal biomass comprises resuspending the previously cultured and dried microalgal biomass in a liquid, such as water. In some embodiments, reconstituting the previously cultured microalgal biomass comprises thawing the previously cultured and frozen biomass, and optionally reconstituting the thawed biomass into a liquid, such as water. Including suspending.

In embodiments described herein, the microalgal biomass has a biomass concentration of about 5% to about 15%, about 3% to about 8%, about 3.5% to about 7.5%, about 4% to about 7%. %, about 4.5% to about 6.5%, or about 5% solids.

In the embodiments described herein, bringing the culture to a certain percent solids content is achieved by, for example, concentrating or diluting the freshly cultured microalgae (e.g., Euglena) with a liquid (e.g., water). can include

In embodiments described herein, the pH of the culture is about 6 to about 11, about 7 to about 10, about 6.5 to about 7.5, about 7, about 8, about 9, about 9.5, about 10, Adjusted to an amount selected from the group consisting of about 10.5, about 11 or about 11.5. In certain embodiments, the pH is adjusted with sodium hydroxide.

In embodiments described herein, the culture is homogenized. In certain embodiments, homogenization is from about 10,000 psi to about 15,000 psi, from about 10,500 psi to about 14,500 psi, from about 11,000 psi to about 14,000 psi, from about 11,500 psi to about 13,500 psi, from about 12,000 psi to about 13,000 psi. Or done at about 12,500psi. In certain embodiments, homogenization is from about 2,000 psi to about 5,000 psi, from about 2,000 psi to about 2,500 psi, from about 3,000 psi to about 3,500 psi, from about 4,000 psi to about 4,500 psi, or from about 5,000 psi to about 5,500 psi. is done in In certain embodiments, homogenization is performed at much lower pressures, such as about 50 psi. In certain embodiments, homogenization is performed in multiple passes.

In the embodiments described herein, homogenization or cell lysis includes one of the following mechanical techniques: high pressure homogenization, bead milling, high shear mixing, pressurized cell disruptor, sonication. or using chemical processes including, but not limited to, enzymatically induced lysis. Homogenization can be performed in the presence of a liquid such as water, solvent or buffered solvent. Homogenization can be performed using a homogenizer such as Polytron PTA-7 and OMNI GLH-01. The homogenizer can be a high pressure homogenizer. In some embodiments, the resuspended biomass is homogenized at about 20° C. to about 27° C. using a high pressure homogenizer at 12,500 psi. Homogenization time depends on the instrument capacity, ie 24 L/h. The collected homogenate is centrifuged at 5,000 rpm for 5 minutes (or 3,500 rpm for 10 minutes) at about 20°C to about 27°C.

In embodiments described herein, the homogenate is centrifuged using a bucket centrifuge at about 3,000 rpm to about 6,000 rpm, about 3,100 rpm to about 5,900 rpm, about 3,200 rpm to about 5,800 rpm, about 3,300 rpm. ~5,700 rpm, 3,400 rpm ~ 5,600 rpm, 3,500 rpm ~ 5,500 rpm, 3,600 rpm ~ 5,400 rpm, 3,700 rpm ~ 5,300 rpm, 3,800 rpm ~ 5,200 rpm, 3,900 rpm ~ approx. A speed selected from the group consisting of 5,100 rpm, from about 4,000 rpm to about 5,000 rpm, from about 4,100 rpm to about 4,900 rpm, from about 4,200 rpm to about 4,800 rpm, from about 4,300 rpm to about 4,700 rpm, or from about 4,400 rpm to about 4,600 rpm. centrifuged at In some embodiments, centrifugation can be performed using a decanter, disc centrifuge, or spiral disc centrifuge.

In embodiments described herein, centrifugation is performed at a g force ranging from about 250 times gravity (xg) to about 16,000xg. In embodiments described herein, the centrifugation is from about 250 x g to about 16,000 x g, from about 500 x g to about 16,000 x g, from about 1000 x g to about 16,000 x g, from about 1,000 x g. Approx. 15,000 x g, Approx. 1,000 x g to Approx. 14,000 x g, Approx. 1,000 x g to Approx. 13,000 x g, Approx. 1,000 x g to Approx. 12,000 x g, Approx. Approx. 10,000 x g, Approx. 1,000 x g to Approx. 9,000 x g, Approx. 1,000 x g to Approx. 8,000 x g, Approx. 1,000 x g to Approx. 7,000 x g, Approx. 1,000 x g to Approx. Ranges from about 5,000×g, from about 1,000×g to about 4,000×g, from about 1,000×g to about 3,000×g, or from about 1,000×g to about 2,000×g.

In embodiments described herein, the homogenate is centrifuged for about 5 minutes to about 30 minutes, about 10 minutes to about 20 minutes, about 13 minutes, or about 15 minutes.

In the embodiments described herein, the centrifuged homogenate is separated into three layers: pellet, middle aqueous layer and top lipid-enriched layer. The pellet is β-glucan (paramylon) and has a white/beige color. The middle aqueous layer contains a soluble protein liquid called protein skim. The top layer is an oil in the form of a waxy emulsion. The three layers are separated and processed separately.

In embodiments described herein, intermediate soluble protein liquids or protein skims are used to produce protein concentrates and/or isolates.

In embodiments described herein, the pH of the protein skim is adjusted to about 3.5 to about 5.5, about 4 to about 5, about 3 to about 5, about 3.5, about 4, about 4.5 or about 5. .

In the embodiments described herein, the pH-adjusted protein skim is dried at 22° C. for at least 1 hour, from about 1 hour to about 24 hours, from about 1 hour to about 12 hours, from about 1 hour to about 6 hours. , about 1 hour to about 4 hours, or about 1 hour to about 2 hours. In embodiments, the pH adjusted protein skim is agitated during incubation.

In embodiments described herein, the incubated and pH adjusted protein skim is centrifuged. In embodiments described herein, centrifugation is performed at about 3,000 rpm to about 6,000 rpm, about 3,100 rpm to about 5,900 rpm, about 3,200 rpm to about 5,800 rpm, about 3,300 rpm to about 5,700 rpm, about 3,400 rpm. ~5,600rpm, ~3,500rpm ~ ~5,500rpm, ~3,600rpm ~ ~5,400rpm, ~3,700rpm ~ ~5,300rpm, ~3,800rpm ~ ~5,200rpm, ~3,900rpm ~ ~5,100rpm, ~4,000rpm ~ ~ The speed is selected from the group consisting of 5,000 rpm, from about 4,100 rpm to about 4,900 rpm, from about 4,200 rpm to about 4,800 rpm, from about 4,300 rpm to about 4,700 rpm, or from about 4,400 rpm to about 4,600 rpm. In embodiments described herein, centrifugation is for about 5 minutes to about 30 minutes, about 10 minutes to about 20 minutes, about 13 minutes, or about 15 minutes.

In embodiments described herein, the incubated and pH adjusted protein skim is separated using filtration.

In aspects described herein, the method of making a protein concentrate further comprises saving the protein sludge from the centrifugation step and discarding the supernatant.

In embodiments described herein, the method of making a protein concentrate further comprises resuspending the protein sludge in an equal weight of water to produce a protein slurry.

In embodiments described herein, the method of making a protein concentrate comprises reducing the pH of the protein slurry from the group consisting of about 5.5 to about 8.5, about 6 to about 8, about 6.5 to about 7.5 or about 7. Further comprising adjusting to the selected amount. In certain embodiments, the pH is adjusted using suitable acids or bases. In certain embodiments, the acid is hydrogen chloride, potassium chloride, acetic acid, adipic acid, carbonic acid, citric acid, fumaric acid, gluconic acid, hydrochloric acid, lactic acid, malic acid, metatartaric acid, phosphoric acid, sulfuric acid, tartaric acid or Selected from the group consisting of any combination. In certain embodiments, the base is ammonium bicarbonate, ammonium carbonate, ammonium citrate, ammonium hydroxide, ammonium phosphate, calcium acetate, calcium carbonate, calcium hydroxide, calcium oxide, calcium phosphate, calcium sulfate, magnesium carbonate, water consisting of magnesium oxide, magnesium oxide, magnesium phosphate, magnesium sulfate, potassium bicarbonate, potassium carbonate, potassium hydroxide, potassium phosphate, sodium bicarbonate, sodium carbonate, sodium hydroxide, sodium phosphate or any combination thereof selected from the group.

In embodiments described herein, the method of making the protein concentrate further comprises spray drying the pH adjusted protein slurry. In the embodiments described herein, spray drying can be accomplished using a spray dryer equipped with either a high pressure atomizer, a two-fluid atomizer, or a centrifugal force atomizer. Alternatively, drying can be accomplished using freeze drying, drum drying or pulse combustion drying.

Chemical Composition of Microalgal Biomass Microalgal biomass produced by the culturing methods described herein comprises microalgal oil and/or protein and the culture medium produced by the microalgae or during fermentation. contains other constituents taken up by microalgae from

In the embodiments described herein, the invention is a whole-cell microalgal biomass containing predominantly intact cells, or containing predominantly or completely lysed cells A microalgal flour is provided which is a homogenate of microalgal biomass. In certain embodiments, the microalgal flour is in powder form and the microalgal biomass comprises at least 40% protein by dry weight and less than 20% triglycerides (oils) by dry weight. In some embodiments, the microalgal biomass comprises at least 20% carbohydrates by dry weight. In some embodiments, the microalgal biomass comprises at least 10% dietary fiber by weight. In some embodiments, the protein is at least 30% to about 45% digestible crude protein.

In some embodiments, the particles have an average size of about 100 μm to about 200 μm. In some embodiments, the particles in the powder have an average size of 180 μm. In some embodiments, the particles have an average size of less than 100 μm. In some embodiments, the particles in the powder have an average size of about 40 μm.

In some embodiments, the powder is formed by pulverizing microalgal biomass to form an emulsion and drying the emulsion. In some embodiments, the microalgal flour has a moisture content of 10% or less by weight.

In some embodiments, the microalgal flour further comprises a food-compatible preservative. In some embodiments, the microalgal flour further comprises a food-compatible antioxidant.

In embodiments described herein, the composition provides a food ingredient comprising said microalgal flour in combination with at least one other protein product suitable for human consumption, wherein the food ingredient comprises dried Contains at least 50% protein by weight. In some embodiments, the at least one other protein product is derived from a plant source. In some instances, the plant source is selected from the group consisting of soybeans, peas, beans, milk, whey, rice, lentils, fava beans, chickpeas and wheat.

In embodiments described herein, the composition comprises microalgal biomass comprising at least 40% protein by dry weight and less than 20% triglycerides (oil) by dry weight, and at least one other edible ingredient. provides a food composition formed by combining with In some embodiments, the at least one other edible ingredient is a meat product. In some embodiments, the food composition is a uncooked product. In some embodiments, the food composition is a cooked product.

In embodiments described herein, the composition provides a food composition formed by combining microalgal biomass comprising at least 13% total dietary fiber by weight and at least one edible ingredient. . In some embodiments, the microalgal biomass comprises from about 13% to about 35% total dietary fiber by weight. In some embodiments, the microalgal biomass comprises about 4% to about 10% soluble fiber. In some embodiments, the microalgal biomass comprises from about 5% to about 25% insoluble fiber.

In some embodiments, the food product contains heterotrophically grown microalgae with reduced chlorophyll content compared to phototrophically grown microalgae. In some embodiments, the microalgal flour has a chlorophyll content of less than 5 ppm, less than 2 ppm, or less than 1 ppm.

Protein-rich microalgal biomass has been produced using different culture methods. Microalgal biomass having a higher percentage of protein content is useful according to the embodiments described herein. The microalgal biomass produced by the culture methods described herein typically contains at least 30% protein by dry cell weight. In some embodiments, the microalgal biomass comprises at least 40%, at least 50%, at least 75% or more protein by dry cell weight. In some embodiments, the microalgal biomass comprises 30% to 75% protein by dry cell weight or 40% to 60% protein by dry cell weight. In some embodiments, the protein in the microalgal biomass comprises at least 40% digestible crude protein. In some embodiments, the protein in the microalgal biomass comprises at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% digestible crude protein. In some embodiments, the protein in the microalgal biomass is 40% to 90% digestible crude protein, 50% to 80% digestible crude protein, or 60% to 75% digestible crude protein. Contains unpurified protein.

In some embodiments, the biomass contains less than 0.01 mg/100 g of selenium. In some embodiments, the biomass comprises from about 20% w/w to about 50% w/w algal polysaccharides. In some embodiments, the biomass comprises at least 15% w/w algal glycoproteins. In some embodiments, the biomass or biomass-derived oil comprises 0 mcg/g to 200 mcg/g, 0 mcg/g to 115 mcg/g, or 50 mcg/g to 115 mcg/g total carotenoids, and in certain embodiments, total carotenoids 20mcg to 70mcg or 50mcg to 60mcg of lutein per gram of content. In some embodiments, the biomass comprises from at least 0.5% to about 10% algal phospholipids. In some embodiments, the biomass or oil derived from algal biomass contains at least 0.10 mg/g, 0.02 mg/g to 0.5 mg/g, or 0.05 mg/g to 0.3 mg/g total tocotrienols, and the specific In embodiments from 0.05 mg/g to 0.25 mg/g is alpha tocotrienol. In some embodiments, the biomass or oil derived from algal biomass contains 0.125 mg/g to 0.35 mg/g total tocotrienols. In some embodiments, the algal biomass-derived oil contains at least 5.0 mg/100 g, 1 mg/100 g to 8 mg/100 g, 2 mg/100 g to 6 mg/100 g, or 3 mg/100 g to 5 mg/100 g of total tocopherols, such as vitamin E. and in certain embodiments from 2 mg/100 g to 6 mg/100 g is alpha tocopherol. In some embodiments, the algal biomass-derived oil contains from 5.0 mg/100 g to 10 mg/100 g tocopherols. In some embodiments, the biomass contains 100 mg to 1000 mg gamma aminobutyric acid (GABA).

In some embodiments, the microalgal biomass comprises 20% to 50% carbohydrates by dry weight. In other embodiments, the biomass comprises 25%-40% or 30-35% carbohydrates by dry weight. Carbohydrates can be dietary fiber and free sugars such as sucrose and glucose. In some embodiments, the free sugars in the microalgal biomass are 1%-10%, 2%-8%, or 3%-6% by dry weight. In certain embodiments, the free sugar component comprises sucrose.

In some embodiments, the microalgal biomass comprises at least 10% soluble fiber. In some embodiments, the microalgal biomass comprises at least 20% to 25% soluble fiber. In some embodiments, the microalgal biomass comprises at least 30% insoluble fiber. In some embodiments, the microalgal biomass comprises at least 50% to at least 70% insoluble fiber. Total dietary fiber is the sum of soluble and insoluble fiber. In some embodiments, the microalgal biomass comprises at least 40% total dietary fiber. In other embodiments, the microalgal biomass comprises at least 50%, at least 55%, at least 60%, at least 75%, at least 80%, at least 90% to at least 95% total dietary fiber.

Processing Microalgal Biomass into Final Food Ingredients The concentrated microalgal biomass produced according to the methods described herein is itself a final food ingredient, with no further or minimal modification. It can be used in foodstuffs with only limited modifications. For example, microalgal biomass can be vacuum packed or frozen. Alternatively, the microalgal biomass can be dried by freeze-drying, a "freeze-drying" process in which the biomass is frozen in a freeze-drying chamber to which a vacuum is applied. Application of vacuum to the freeze-drying chamber results in sublimation (primary drying) and desorption (secondary drying) of water from the biomass. However, the present disclosure provides a variety of microalgae-derived final food ingredients with enhanced properties.

The microalgal biomass, microalgal flours, protein concentrates and protein isolates described herein provide improved functionality over conventional plant proteins, and the microalgal biomass is derived from microalgal of biomass does not increase the viscosity of the food to which it is added or blended. In some embodiments, the food product has a viscosity of from about 1 mPa·s to about 2000 mPa·s at 25°C.

The microalgal biomass, microalgal flour, protein concentrates and protein isolates described herein have good foaming properties.

In some embodiments, the compositions are formulated into swallowable, chewable, or dissolvable oral dosage forms. Swallowable compositions are well known in the art and are compositions that do not immediately dissolve when placed in the mouth and can be swallowed whole without any chewing.

To prepare a swallowable composition, the microalgal biomass (wet or dry) can be combined with a suitable carrier (e.g., excipients, stabilizers, binders, etc.) according to conventional compounding techniques. In some embodiments, the swallowable composition can be coated with a polymeric film. Such film coatings have several beneficial effects. First, such a film coating reduces adhesion of the composition to the inner surfaces of the mouth, thereby increasing the ability to swallow the composition. Second, the film can help mask the unpleasant taste of certain ingredients. Third, film coatings can protect the compositions of the present invention from atmospheric degradation. Polymeric films that can be used in preparing the swallowable composition include vinyl polymers such as polyvinylpyrrolidone, polyvinyl alcohol and acetate, celluloses such as methyl and ethyl cellulose, hydroxyethyl cellulose and hydroxypropylmethyl cellulose, acrylates and methacrylates, vinyl -copolymers such as maleic acid and styrene-maleic acid types, and natural gums and resins such as zein, gelatin, shellac and gum arabic.

A chewable composition is a composition that has a palatable taste and texture, is relatively soft, and rapidly breaks down into smaller pieces and begins to dissolve after chewing such that it is swallowed substantially as a solution. .

To create a chewable composition, certain ingredients should be included to achieve the attributes just described. For example, chewable compositions should contain ingredients that create a pleasant flavor and mouthfeel and promote relative softness and solubility in the mouth. The following description describes ingredients that can help achieve these characteristics.

A chewable composition should begin to disintegrate and dissolve in the mouth shortly after chewing begins so that the composition can be swallowed substantially as a solution. The dissolution profile of the chewable composition can be enhanced by the inclusion of fillers and excipients that dissolve rapidly in water. Fillers and excipients that dissolve rapidly in water preferably dissolve within about 60 seconds of wetting with saliva. In fact, it is believed that if sufficient water-soluble excipients are included in the composition of the present invention, the composition may become dissolvable rather than chewable composition form. Examples of rapidly water-dissolving fillers suitable for use with the present invention include, but are not limited to, sugars, amino acids, and the like. A disintegrant may also be included in the composition of the present invention to facilitate dissolution. Disintegrants, including permeabilizing agents and wicking agents, can draw water or saliva into the composition, thereby facilitating dissolution of the composition from the inside and outside. Such disintegrants, permeabilizers and/or water-conducting disintegrants that may be used in the present invention include, by way of example, starches such as corn starch, potato starch, pregelatinized and modified starches thereof, cellulosic Active substances such as Ac-di-sol, montmorillonite clay, crosslinked PVP, sweeteners, bentonite, microcrystalline cellulose, croscarmellose sodium, alginates, sodium starch glycolate, gums such as agar, guar, locust bean, karaya , pectin, arabic, xanthan and tragacanth, silica with high affinity for aqueous solvents such as colloidal silica, precipitated silica, maltodextrin, β-cyclodextrin, polymers such as carbopol, and cellulosic active substances such as Examples include, but are not limited to, hydroxymethylcellulose, hydroxypropylcellulose and hydroxypropylmethylcellulose.

In embodiments described herein, the microalgal biomass composition can be formulated in the form of a liquid gelatin capsule. This may comprise microalgal biomass suspended, dissolved or contained in a suitable liquid vehicle encapsulated in a gelatin shell, generally comprising gelatin with a plasticizer such as glycerin or sorbitol. Filler materials may include, for example, polyethylene glycol.

Microalgal Flour The protein content of the microalgal flour can vary depending on the percent protein of the microalgal biomass. Microalgal flour can be produced from microalgal biomass of varying protein content. In certain embodiments, the microalgal flour is produced from microalgal biomass of the same protein content. In some embodiments, the microalgal flour is produced from microalgal biomass of different protein content. In the latter case, microalgal biomass of varying protein content can be combined, followed by a homogenization step. In other embodiments, microalgal flours of varying protein content are first produced and then combined in varying proportions to achieve a microalgal flour product containing the final desired protein content. blended. In certain embodiments, microalgal biomass of different protein profiles can be combined together and then homogenized to produce microalgal flour. In another embodiment, microalgal flours of different protein profiles are first produced and then blended together in varying proportions to achieve a microalgal flour product containing the final desired protein profile. .

The microalgal flours described herein are useful in a wide variety of food preparations. Due to the protein content, fiber content and micronized particles, microalgal flour is a multifunctional food ingredient. Microalgal flour can be used in baked goods, quick breads, yeast dough products, egg products, dressings, sauces, nutritional drinks, algal milk, pasta and gluten-free products. Gluten-free products include microalgae flour, amaranth flour, arrowroot flour, buckwheat flour, rice flour, chickpea flour, cornmeal, maize flour, millet flour, potato flour, potato starch flour, and quinoa flour. , Sorghum Flour, Soybean Flour, Bean Flour, Legume Flour, Tapioca Flour, Teff Flour, Artichoke Flour, Almond Flour, Acorn Flour, Coconut Flour, Chestnut Flour, Corn Flour , can be made using another gluten-free product such as taro flour. Microalgae flour in combination with other gluten-free ingredients is useful in producing gluten-free foods such as baked goods (cakes, cookies, brownies and cake-like products (e.g. muffins)), breads, cereals, crackers and pasta. is useful in Further details of incorporating microalgal flour into these foods and the like are provided in the Examples below.

Microalgal flour can be used in baked goods in place of traditional protein sources (eg, nuts, meat products or beans) and eggs. Baked foods and gluten-free products have excellent moisture content and a crumb structure indistinguishable from conventional baked foods made with butter and eggs. Due to their superior moisture content, these baked foods have a longer shelf life than traditional baked foods produced without microalgae flour, preserving the original texture of these baked foods. hold longer.

Water activity (Aw) of food can be an indicator of shelf-life retention in cooked foods. Water activity (ranging from 0 to 1) is a measure of how efficiently water present in food can participate in chemical or physical reactions. The water activities of some common foods exhibiting a spectrum of Aw are fresh fruit/meat/milk (1.0-0.95); cheese (0.95-0.90); margarine (0.9-0.85); nuts (0.75-0.65). cured meats (0.85-0.80); jams (0.8-7.5); pasta (0.5); cookies (0.3); and dried vegetables/crackers (0.2). Most bacteria do not grow at water activities below 0.91. Below 0.80 most molds cannot grow, and below 0.60 no microbial growth is possible. By measuring water activity, it is possible to predict potential sources of spoilage. Water activity can also play an important role in determining the activity of enzymes and vitamins in foods that can greatly affect the color, taste and aroma of foods.

Microalgae flour can also act as a protein supplement for use in smoothies, sauces or dressings.

Microalgal flour can also be added to powdered or liquid eggs that are commonly served in food service situations. The combination of the powdered egg product and the microalgal flour is itself a powder that can be combined with an edible liquid or other edible ingredients and typically then cooked to form a food product. In some embodiments, the microalgal flour is then spray dried to form a powdered food ingredient (e.g., powdered eggs, powdered sauce mixes, powdered soup mixes, etc.). can be combined with any liquid product In such cases, after homogenization, but before drying, the microalgal flour is combined with the liquid product, as in a slurry or dispersion, and then the combination is spray dried to produce a powdered food ingredient. It is advantageous to form This co-drying process increases the homogeneity of the powdered food ingredient compared to mixing the dried forms of the two ingredients together. The addition of microalgae flour improves the appearance, texture and mouthfeel of powdered and liquid eggs, even when the cooked eggs are held on a steam table, resulting in improved appearance, texture. and long-lasting mouthfeel.

The microalgal flour can be used to formulate a reconstituted food product by combining the microalgal flour with one or more edible ingredients and a liquid, such as water. Reconstituted food products can be beverages, dressings (such as salad dressings), sauces (such as cheese sauce), or intermediates such as dough that can then be baked. In some embodiments, the reconstituted food product is then subjected to shear forces such as pressure breaking or homogenization. The particle size of the preferred microalgal flour in the reconstituted food product averages 1-15 micrometers.

Combining Microalgal Biomass or Materials Derived from the Biomass with Other Food Ingredients The compositions described herein containing microalgal biomass are formulated for consumption as, for example, a meal, a nutraceutical or a dietary supplement. be done. In some embodiments, the composition comprising microalgal biomass is in solid, powder or liquid form and formulated for oral administration. In another aspect, the composition comprising microalgal biomass is formulated as a food additive.

In some embodiments, the composition is formulated as a food additive and added to food. For example, without limitation, the compositions may include sauces, teas, candies, cookies, cereals, breads, fruit mixes, fruit salads, salads, snack bars, protein bars, fruit leathers, yogurts, health bars, granola, smoothies, soups, It can be added to juices, cakes, pies, shakes, ice creams, protein drinks, nutritional drinks, nutritional drink supplements, animal analogs and health drinks. Compositions formulated as food additives can have any of the additives described above for oral formulations.

In some embodiments, the composition is formulated as a non-dairy product such as cheese or yogurt.

In some embodiments, the composition is formulated as an animal analogue. In embodiments, the animal analogue is a meat analogue. In some embodiments, the animal analogue is an egg analogue. In some embodiments, the egg analogue is in liquid form. In some embodiments, the egg analogue is in dried or powdered form. In some embodiments, the animal analogue is a meat analogue, a sausage analogue, a pepperoni/cured meat analogue, a chicken analogue, a turkey analogue, a pork analogue, a bacon analogue, a beef analogue, a tofu substitute. , ground beef analogues, jerky analogues, egg analogues, egg replacers, hard-boiled egg replacers, powdered egg replacers, liquid egg replacers, frozen egg replacers, salad dressings, mayonnaise and combinations thereof is selected from

In some embodiments, the composition is formulated as an extruded product such as protein crisps.

In some embodiments, the composition is formulated as a nutritional drink or nutritional drink supplement. In some embodiments, the nutritional beverage is in liquid form. In some embodiments, the nutritional beverage is in powdered form.

In some embodiments, the composition is formulated as a protein bar.

Any of the compositions described herein can also be formulated as foods.

In some embodiments, the composition is in liquid form. In some embodiments, the composition is in powdered form.

In some embodiments, the microalgal biomass is combined with other ingredients (eg, masking substances, flavorants and/or other additional ingredients). Such other ingredients may be added at any time during the cultivation or processing of the microalgal biomass, or may be added directly to the final or intermediate food product. For example, such other components may be spray dried during culturing, onto the wet biomass prior to drying (e.g., spray drying), during drying (e.g., spray drying), onto the wet biomass, during spray drying. It can be applied to ingredients, to finished foods, or any combination thereof. As discussed herein, wet biomass can be used directly in food preparation. Accordingly, compositions and food products according to some embodiments include additional ingredients including flavorants, masking agents and/or additional ingredients. Methods according to some embodiments include applying flavorants, masking agents and/or additional ingredients to culturing microalgae and/or microalgal biomass.

Typically, the composition contains about 0.01% to about 100%, about 0.01% to about 99%, about 0.01% to about 95%, about 0.01% to about 80%, about 0.01% to about 75%, about 0.1% to about 100%, about 0.1% to about 99%, about 0.1% to about 95%, about 0.1% to about 80%, about 0.1% to about 75%, about 1% to about 100%, about 1% ~ about 99%, about 1% to about 95%, about 1% to about 80%, about 1% to about 75%, about 5% to about 100%, about 5% to about 99%, about 5% to about 95%, about 5% to about 80%, about 5% to about 75%, about 10% to about 100%, about 10% to about 99%, about 10% to about 95%, about 10% to about 80% , containing from about 10% to about 75% microalgal biomass.

In some embodiments, the food composition comprises at least 0.1% w/w microalgal biomass and one or more other edible ingredients. In some embodiments, the food composition comprising microalgal biomass comprises at least 10% protein by dry weight. In some embodiments, the microalgal biomass contains about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or 60% protein by dry weight. do. In some embodiments, the microalgal biomass contains 10% to 90%, 10% to 75%, 25% to 75%, 40% to 75%, or 50% to 70% protein by dry weight. . In a preferred embodiment, the microalgal biomass is grown under heterotrophic conditions and has reduced green pigmentation.

In embodiments described herein, the food composition comprises at least 0.1% w/w microalgal biomass and one or more other edible ingredients, wherein the microalgal biomass is dried at least 30% protein by weight, at least 40% protein by dry weight, at least 45% protein by dry weight, at least 50% protein by dry weight, at least 55% protein by dry weight, at least 60% by dry weight of protein or at least 75% protein by dry weight. In some embodiments, the algal biomass contains 30% to 75% or 40% to 60% protein by dry weight. In some embodiments, at least 40% of the unpurified protein is digestible, at least 50% of the unpurified protein is digestible, at least 60% of the unpurified protein is digestible, and at least 70% digestible, at least 80% of the crude protein is digestible, or at least 90% of the crude protein is digestible. In some embodiments, the microalgal biomass is grown under heterotrophic conditions. In some embodiments, the microalgal biomass is grown under nitrogen-rich conditions.

In the embodiments described herein, the microalgal biomass comprises predominantly intact cells. In some embodiments, the food composition comprises oil predominantly or completely encapsulated within the cells of the microalgal biomass. In some embodiments, the food composition comprises predominantly intact microalgal cells. In some embodiments, the microalgal oil is primarily encapsulated within the cells of the biomass. In some embodiments, the microalgal biomass comprises predominantly lysed cells (eg, homogenates). As noted above, such homogenates can be provided as slurries, flakes, powders or powders.

In the embodiments described herein, the food composition comprises one or more other edible ingredients including, but not limited to grains, fruits, vegetables, proteins, lipids, herbal and/or spice ingredients. containing microalgal biomass combined with In some embodiments, the food composition is a salad dressing, egg product, baked goods, bread, bar, pasta, sauce, soup drink, beverage, frozen dessert, butter or spread. In some embodiments, the food composition is not a pill or powder. In some embodiments, the weight of the food composition is at least 50g, or at least 100g.

The microalgal biomass can be combined with one or more other edible ingredients to produce a food product. The microalgal biomass can be microalgal biomass from a single algal source (eg, strain) or multiple sources (eg, different strains). Biomass can also be derived from a single algal species, but with different composition profiles. For example, a manufacturer can blend high oil content microalgae with high protein content microalgae to achieve the exact oil and protein content desired in the final food product. The combination can be performed by food manufacturers to produce finished products for retail or food service. Alternatively, the manufacturer can market the microalgal biomass as a product and the consumer can incorporate the microalgal biomass into food products, eg, by modification of conventional recipes. In either case, algal biomass is typically used to replace all or part of the proteins, oils, fats, eggs, etc. used in many conventional foods.

In some embodiments, the food composition formed by the combination of microalgal biomass and/or products derived therefrom, i.e. microalgal flour, protein concentrate or protein isolate, contains at least 0.1% w/w or v/v, at least 0.5% w/w or v/v, at least 1% w/w or v/v, at least 5% w/w or v/v, at least 10% w/w or v/v, at least 25% w/w or v/v or at least 50% w/w or v/v of microalgal biomass. In some embodiments, the formed food composition contains at least 2% w/w, at least 5% w/w, at least 10% w/w, at least 25% w/w, at least 50% w/w, at least 75% w/w, at least 90% w/w or at least 95% w/w of microalgal biomass or products derived therefrom. In some cases, the food composition comprises 5% to 50%, 10% to 40%, or 15% to 35% by weight or volume of algal biomass or products derived therefrom.

Microalgal biomass, including microalgal flour, protein concentrates or protein isolates, can be incorporated into virtually any food composition. Some examples include baked foods such as cakes, brownies, yellow cakes, breads including brioche, cookies including sugar cookies, biscuits and pies. Other examples include products often provided in dried form, such as pasta or powdered dressings, dried creamers, minced meat and meat substitutes. Incorporation of predominantly intact microalgal biomass as a binder and/or bulking agent in such products can improve hydration and increase yields, primarily due to the water binding capacity of the intact biomass. . Foods that are rehydrated, such as scrambled eggs made from dry powdered eggs, can also have improved texture and nutritional profile. Other examples include liquid foods such as sauces, soups, dressings (ready to eat), creamers, milk drinks, juice drinks, smoothies, creamers, and the like. Other liquid foods include nutritional drinks that serve as meal replacements or algal milks. Other food products include butter or cheese, including shortening, margarine/spreads, nut butters and cheese products such as nacho sauces. Other food products include energy bars, chocolate confectionery-lecithin substitutes, meal replacement bars and granola bar type products. Another type of food is batters and coatings. By providing a layer of oil surrounding the food, the predominantly intact biomass or homogenate repels additional oil from the cooking medium and prevents it from penetrating the food. Thus, the food product can retain the benefits of the high monounsaturated oil content of the batter without picking up less desirable oils (eg, trans fats, saturated fats and by-products from cooking oils). Biomass batter can also provide a desirable (eg, crunchy) texture and cleaner flavor to the food product because less cooking oil and its by-products are absorbed.

In certain embodiments, the protein food bar is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, About 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95 % contains Euglena powder.

In certain embodiments, the protein food bar contains about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35% Euglena until the desired texture is achieved. Contains beta-glucan isolate.

In certain embodiments, the protein bar or food product comprises about 20% to about 35% dates, about 10% to about 15% agave, about 5% to about 15% natural peanut butter, About 1% to about 5% coconut oil, about 5% to about 15% rolled oats, about 5% to about 15% almonds, and about 5% to about 40% euglena flour and about 0% to Contains approximately 10% beta-glucan isolate.

In certain embodiments, the protein bar or food product comprises almonds, dates, agave, natural peanut butter, coconut oil, rolled oats, euglena flour, maple syrup, beta-glucan isolate (BGI), ready-to-gel ( RTG (Ready-to-gel), beta-glucan powder, RTG wet gel, tapioca syrup, agave syrup, Golden strap Molasses, flavor masking, syrups and combinations thereof.

In certain embodiments, the materials required in forming a protein bar food product are cooking scales, spatulas, spoons, food processors, cooking ranges, double boilers, mixing bowls, wax paper , bar molds and combinations thereof.

In certain embodiments, the cooking range heats to about 330°F to about 380°F. The articles are combined to create a flexible fabric. Once the dough is formed, a portion of the dough is pressed into the mold at the desired level using suitable cooking equipment. Once the mold is filled, wrap the product in food preservative material and place in the refrigerator to harden.

In uncooked food, most algal cells in the microalgal biomass remain intact. This has the advantage of protecting the algal oil from oxidation, giving it a long shelf life and minimizing detrimental interactions with other ingredients. Depending on the nature of the food, the protection conferred by cells may reduce or avoid the need for refrigeration, vacuum packaging, and the like. Leaving the cells intact also prevents direct contact between the oil and the consumer's mouth, reducing the oily or fatty sensation that may be undesirable. In foods where oil is used more as a dietary supplement, this can be an advantage in improving the organoleptic properties of the product. Therefore, predominantly intact microalgal biomass is suitable for use in such products. However, in uncooked products such as salad dressings where the oil (e.g., as an emulsion with an aqueous solution such as vinegar) provides the desired mouthfeel, the use of refined algal oil or micronized biomass is preferred. In cooked foods, some algal cells of the original intact microalgal biomass may be lysed while other algal cells may remain intact. The ratio of lysed to intact cells depends on the temperature and duration of the cooking process. In cooked foods (e.g. baked foods) where distribution of the oil in a uniform manner with other ingredients is desired for taste, texture and/or appearance, micronized biomass or refined The use of algal oil is preferred. In cooked foods, microalgal biomass is used to supply oil and/or protein and other nutrients primarily because of the nutritional or caloric value of the biomass rather than its texture.

Microalgal biomass, including microalgal flour, protein concentrates or protein isolates, can also be manufactured into nutritional or dietary supplements. For example, microalgal flour, protein concentrates or protein isolates can be encapsulated in digestible capsules in a manner similar to other dietary supplements. Such capsules may be packaged in bottles and taken daily (eg, 1-4 capsules or tablets per day). The capsule can contain a unit dose of microalgal flour, protein concentrate or protein isolate. Similarly, the microalgal biomass can optionally be compressed into tablets with pharmaceuticals or other excipients. Tablets may be packaged, eg, in bottles or blister packs, and taken daily, eg, in doses of 1 to 4 tablets per day. In some cases, a tablet or other dosage formulation contains a unit dose of microalgal flour, protein concentrate, or protein isolate. Capsule and tablet products and other food supplements are preferably manufactured under GMP conditions suitable for dietary supplements as codified in Title 21 Part 111 of the Code of Federal Regulations, United States Code of Federal Regulations, or equivalent regulations enacted by foreign jurisdictions. is done in The microalgal flour, protein concentrate or protein isolate is mixed with other powders and packaged in a sachet as a ready-to-mix material (e.g. with water, juice, milk or other liquids). can be put in and served. Algal biomass can also be incorporated into products such as yogurt.

Microalgal biomass, including microalgal flour, protein concentrate or protein isolate, is packaged in a form in which it is combined with other dry ingredients (e.g. sugar, flour, dried fruit, flavorings) and is used in the final product. It can also be portioned packs to ensure uniformity. The mixture is then converted into food by the consumer or food service company simply by adding liquids such as water or milk, optionally mixing, and/or cooking without the addition of oil or fat. obtain. In some embodiments, a liquid is added to reconstitute the dried microalgal biomass composition. Cooking can optionally be done using a microwave, convection oven, conventional oven, or on a countertop. Such mixtures can be used to make cakes, breads, pancakes, waffles, beverages, sauces, and the like. Such mixtures have the advantages of consumer convenience and long shelf life without refrigeration. Such mixtures are typically packaged in a sealed container with instructions for adding liquids to transform the mixture into a food product.

Example 1: Regulation of protein content Protein content is controlled primarily by the C:N ratio. A lower C:N ratio results in a higher protein content. Adjustment of the lipid content focuses on the dissolved oxygen content of the reactor. Under hypoxic/anaerobic conditions, Euglena converts paramylon to wax esters. Table 1 gives the amount of protein or β-glucan produced under different C:N ratios in the culture medium.

(Table 1) C:N ratio of biomass focused on protein or β-glucan

Example 2: Nutrient Contents of Euglena Based on publicly available reported studies, the nutrient contents of up to 59 reported nutrients of Euglena are provided herein. The nutritional complexity of Euglena can be thought of in terms of 'healthy' or 'total nutritional care', with the powder instead of individual ingredients providing this holistic benefit. The nutritional value of Euglena flour depends on environmental conditions.

Approximate and other nutrients are shown in Table 2 below. Cholesterol levels were below the limit of detection. Regarding GABA, Euglena protein powder contains 417mg/100g of GABA. This means that the protein-enriched dried biomass of Euglena, which is within the dosage range of some anxiety medications, has additional health properties beyond just basic macronutrients.

(Table 2) Nutrient profile from 100 g of Euglena protein powder. ND stands for not detected.

The fatty acid profile for 6.1% fat measured in a 100 g Euglena protein flour sample is shown in Table 3 below. Notably, trans fatty acids were below the limit of detection. About half of the fatty acids were saturated fatty acids. About 20% polyunsaturated fatty acids such as omega 3, 5 and 9 were also present in the biomass. This gives the flour some nutritional properties without being a high fat product.

脂肪酸は、粉中の脂肪酸（FA）のパーセンテージ、総脂質中のFAのパーセンテージ、および総粉100g当たりのmgでの質量で報告されている。 (Table 3) Fatty acid profile of Euglena protein powder

Fatty acids are reported as percentage of fatty acids (FA) in flour, percentage of FA in total lipid, and mass in mg per 100 g of total flour.

The amino acid profile of the protein flour is shown in Table 4 below. A 100g serving of Euglena protein powder contains a total of 27.4g of protein.

(Table 4) Amino acid profile in 100 g of Euglena protein powder

Both minerals (Table 5) and vitamins (Table 6) have over 10% of the daily intake in 100g of powder. This provides additional nutrition to products incorporating the Euglena protein powder.

(Table 5) Mineral profile in 100 g of Euglena protein powder

N/Aは、該当なしを表す。 (Table 6) Vitamin profile in 100 g of Euglena protein powder

N/A indicates not applicable.

Example 3: Euglena Protein Concentrate Methods and Examples Case 1: Two-Phase Protein Production (HS Protein Concentrate and LS Protein Concentrate)
Frozen biomass from the harvest was thawed and blended in a commercial blender for 10 minutes. The biomass was diluted with water to approximately 5% solids. The diluted biomass was then neutralized with 1.5 molar sodium hydroxide and then homogenized in a single pass at 12,000 psi in an SPX APV 1000 Lab Series Homogenizer. The homogenate was centrifuged (4700 rpm for 13 minutes) to obtain a light phase of dilute high solubility (HS) proteins called HS skim phase and low solubility (LS) proteins mixed with paramylon (PM) called LS/PM sludge. A two-phase system with heavy-phase sludge was obtained.

The HS skim was collected by decantation, then the skim was adjusted to pH 4.5 with 50% w/w citric acid in water, followed by gentle intermittent agitation for 1 hour followed by centrifugation at 4,700 rpm for 13 minutes. . HS skim precipitation yielded two phases, a light phase called HS whey (a soluble cellular component that did not precipitate under acid) and HS sludge. The HS sludge was then diluted with an equal mass of water, neutralized with 1.5 M NaOH, and then lyophilized to obtain a powdered HS protein concentrate. HS whey was discarded. After lyophilization, the HS protein concentrate powder was analyzed by proximity analysis at SGS Mississauga.

To obtain the LS protein concentrate powder, the LS/PM sludge was diluted with an equal volume of water and adjusted to pH 10 with 1.5M NaOH while blending in a commercial blender for approximately 10 minutes. Following this alkaline solubilization step, the mixture was centrifuged at 4,700 rpm for 13 minutes to obtain two phases, a light phase consisting of solubilized low solubility protein (LS skim) and a heavy phase consisting of paramylon sludge. The LS skim was decanted prior to protein precipitation by adjusting the pH to 4.5 with 50% w/w aqueous citric acid. After 1 hour at pH 4.5 with gentle agitation, the LS skim was centrifuged at 4,700 rpm for 13 minutes to obtain a light phase of acid-soluble cellular material (LS whey) and a precipitate of low-soluble proteins called LS sludge. rice field. After diluting the LS sludge with an equal mass of water, it was neutralized with 1.5M NaOH and lyophilized to obtain the LS protein concentrate powder, which was also subjected to approximate analysis as shown in Table 7.

Table 7 Approximate Data for Biphasic Protein Production of Case 1

Case 2: Alkali separation after homogenization Frozen biomass from the harvest was thawed and blended in a commercial blender for 10 minutes. The biomass was diluted with water to approximately 5% solids. The diluted biomass was then neutralized with 1.5 molar sodium hydroxide and then homogenized in a single pass at 12,000 psi in an SPX APV 1000 Lab Series Homogenizer. The homogenate was immediately adjusted to pH 10 with 1.5M NaOH. The pH-adjusted homogenate was gently agitated for 1 hour to ensure maximum protein solubilization obtained under these conditions. After agitation, the homogenate was centrifuged at 4,700 rpm for 13 minutes.

After centrifugation only two phases were obtained, a heavy phase consisting mostly of paramylon (PM sludge) and a light phase containing dissolved proteins and any other weakly alkaline soluble cellular components (PC skim). The PC skim phase was then adjusted to pH 4.5 using 50% w/w aqueous citric acid. After pH adjustment, the mixture was gently stirred overnight (approximately 16 hours) and then centrifuged at 4,700 rpm for 13 minutes. This separation yielded a light phase consisting of alkali- and acid-soluble cellular components (PC whey) and a heavy sludge phase containing precipitated proteins (PC sludge). The PC sludge was then diluted with an equal mass of water, neutralized with 1.5 M NaOH, and then spray dried using a LabPlant SD-06 spray dryer at an inlet temperature of 160 C and a feed rate of approximately 10 mL/min. did. The resulting powdered protein concentrate was sent for proximity analysis at SGS-Mississauga as shown in Table 8.

Table 8 Approximate data from post-homogenization alkaline separation of Case 2

Case 3: Alkaline extraction before homogenization
TK4300 biomass from two harvests was thawed from freezing, combined and diluted with water to 5% solids. The diluted biomass blend was then mixed in a stirred kettle for 15 minutes. After resuspension and mixing, the biomass was adjusted to pH 10 using 1.5M NaOH. After pH adjustment, the biomass was then homogenized in an SPX APV 1000 Lab Series homogenizer at 12,000 psi. The resulting homogenate was then centrifuged at 4,700 rpm for 13 minutes to separate a light phase containing solubilized proteins and other alkali-soluble cellular components (PC skim) and a heavy phase consisting mainly of paramylon (PM sludge). obtained two phases of The PC skim was adjusted to pH 4.5 using 85% w/w phosphoric acid followed by gentle stirring for 1 hour to complete precipitation. After the precipitation reaction, the PC skim was centrifuged at 4,700 rpm for 13 minutes to obtain two phases, a light phase consisting of acidic and alkaline soluble components (whey) and a heavy phase consisting of precipitated proteins (PC sludge).

PC sludge was diluted to 10% solids, adjusted to pH 7 with 1.5M NaOH, and run in a 35 inch Wild Horse International Spray Dryer, with variable flow rates to maintain an inlet temperature of 250C and an outlet temperature of 70-75C. Spray dried using S/S, Model LPG-5. The resulting protein concentrate powder was sent for proximate analysis at SGS Mississauga as shown in Table 9.

Table 9. Approximate data from pre-homogenization alkaline extraction of Case 3

Case 4: Acid homogenization process
TK4300 biomass from two harvests was thawed from freezing, combined and diluted with water to 5% solids. The diluted biomass blend was then mixed in a stirred kettle for 15 minutes. Diluted biomass was adjusted to pH 4.7 using 1.5 M NaOH (starting at pH 3.3). The biomass was then homogenized at 12,000 psi in an SPX APV 1000 Lab Series Homogenizer. The homogenate was stored overnight in a 4°C refrigerator. The next day, the homogenate was centrifuged at 4,700 rpm for 13 minutes to obtain a three-phase mixture. The lightest phase contains acid-soluble cellular components (whey) and the resulting sludge consists mainly of the upper part composed mainly of precipitated proteins (PC sludge) and the denser paramylon (PM sludge). Two slightly separate phases formed with a horizontal line separating the bottom and bottom. Whey was decanted and discarded. The PC sludge was then manually removed from the PM sludge using a hand tool in a centrifuge bottle and collected. PM sludge was processed separately into β-glucan isolates.

The PC sludge was then diluted to 10% solids, adjusted to pH 7 and dried in a 35 inch Wild Horse International Spray Dryer, S/S, with variable flow rates to maintain an inlet temperature of 250C and an outlet temperature of 70-75C. Spray dried using Model LPG-5. Protein concentrate powders were collected and sent for proximate analysis at SGS Mississauga as shown in Table 10.

Table 10 Approximate data from the acidic homogenization process of Example 4

Case 5: In-process future optimization example for protein concentrates and isolates The example above relies on multiple key unit operations that can be further optimized. . Operations that warrant further optimization include, but are not limited to, homogenization, precipitation, phase separation and drying.

Homogenization (and alternative cell lysis techniques):
Homogenization unit operations include, but are not limited to, biomass solids content, homogenization temperature, number of passes, homogenization pressure and homogenization valve geometry and number of stages. It has multiple parameters that are not optimized. For example, altering the solids content of homogenization can affect both the effectiveness of lysis and whether certain biomolecules associate in a concentration-dependent manner (ie, proteins and lipids). Optimizing temperature not only further influences the thermodynamics of biomolecule separations, but also potentially limits thermal denaturation of proteins, resulting in more highly functional concentrates and isolates. The number of homogenizer passes as pressure and geometry all directly affect the separation of protein from lipids, as excessive homogenization results in emulsification that dramatically adversely affects protein and lipid separation. . Minimizing lipid emulsification through gentler dissolution is expected to result in higher protein content concentrates and/or isolates. Additionally, alternative cell lysis techniques including, but not limited to, enzymatic lysis, ultrasonic lysis, chemical lysis or milling may be utilized to achieve the same effect.

Precipitation:
Precipitation procedures can be influenced by the choice of acid or other precipitant used, the concentrations of acids and bases used for conditioning, the agitation mechanism and speed, the temperature and time used for the reaction. Different acids, through different chemical structures, can interact differently with proteins, altering protein structure and thus solubility and functionality. Further research has shown that different acids as well as other non-acid precipitators such as ammonium sulfate or other food-safe salts belonging to the Hoffmeister family can be used to salt out proteins from solution. would consider drugs. Agitation mechanisms and speeds can significantly affect protein solubility depending on whether they introduce air or not. The introduction of air creates an air-liquid interface at which the protein is forced to change its structure, which ultimately alters the ultimate functional properties of the protein upon recovery. can let Optimal agitation mechanisms are selected so as not to perturb the native protein structure and maintain optimal functionality. The time of reaction determines the yield and amount of contaminants co-purified during subsequent unit operations. Alternatively, precipitation is avoided by selectively removing other contaminants such as lipids and carbohydrates using chromatographic techniques that selectively adsorb either proteins or impurities prior to the drying process. obtain.

Isoelectric precipitation:
The clarified product can be transferred to tanks equipped with pH, temperature and agitation control sensors. The pH can be adjusted to promote isoelectric precipitation under controlled conditions such as protein concentration, stirring time, temperature and shear rate. Isoelectric focusing can be performed in different steps to precipitate maximum soluble protein from the centrifugate. Additionally, heat treatment may be applied to accelerate the precipitation process. Subsequently, continuous centrifugation (separator plate centrifuge Separation) can recover the precipitate.

Alternatively, the sediment can be separated through a sedimentation process in a sedimentation tank. Additionally, the precipitation process can be accelerated by using food grade flocculants.

Phase separation:
The phase separation operation depends on the centrifuge technology (disc-stack, decanter, tubular bowl, etc.), centrifuge parameters (feed rate, speed, weir, etc.). It presents many options for future optimizations, such as fixing the depth of Different centrifuges and operating parameters allow modification of the applied force that affects the mechanical separation of precipitated proteins from the surrounding aqueous environment, which ultimately leads to contaminants such as lipids and carbohydrates. Better exclusion results in higher protein content in the produced concentrate. Alternatively, various phase separation techniques other than centrifugation will be tested, including but not limited to ultrafiltration and chemically induced phase partitioning. Ultrafiltration can be used to concentrate the protein from the lysate and may not require the use of any precipitant, only achieving a higher protein content in the concentrate. instead, the functionality of the final product can be improved. Chemical phase partitioning can be accomplished by adding a water-soluble polymer such as polyethylene glycol to the system, which forms a multiphasic aqueous mixture in which there are polymer-rich and polymer-poor phases. Proteins are also selective to these phases, such that simple decantation or accelerated mechanical separation of the newly formed phases can then separate the proteins from other cellular components, followed by separation of the proteins from the polymer. distributed to

Membrane filtration:
Membrane filtration is a widely used separation technique in biological processing. Depending on the porosity of the membrane, membrane filtration can be classified as a microfiltration or ultrafiltration process. Microfiltration membranes are commonly used for clarification, sterilization and particulate removal or for cell separation. Ultrafiltration membranes are commonly used for concentration and desalting, buffer exchange and fractionation of dissolved molecules (proteins, peptides, nucleic acids, etc.). Two common modes of membrane process operation are dead end (dead end filtration) and cross flow (tangential flow filtration) filtration modes. In cross-flow filtration mode, the pressure differential across the membrane causes the fluid to be filtered to flow parallel to the membrane surface to retain high molecular weight molecules and pass water and low molecular weight solutes into the filtered product. Cross-flow reduces filter cake formation and keeps filter cake at a low level. Two major variables, the transmembrane pressure TMP and the cross-flow or feed flow rate, are controlled in all tangential flow devices during UF/DF operation.

Ultrafiltration can be used to concentrate soluble proteins from the centrate. The correct molecular weight cut-off (MWCO) membrane used to retain maximum protein can be selected based on screening experiments. The operating process parameters can then be optimized to achieve maximum flow. Additionally, rounds of diafiltration can be performed to achieve higher protein content or to create protein isolates.

drying:
Drying operations offer another opportunity to optimize both the purity and functionality of protein concentrates and/or isolates. Purity can be affected by using air flotation or triboelectric separation at the same time as the dryer, which can separate proteins from other components based on density and charge, respectively. Final product functionality is significantly affected by the drying technique, and optimization of this procedure is essential for highly functional protein products. Proteins are readily heat denatured, resulting in changes in protein conformation in response to heat from the protein's environment. Most drying techniques utilize heat and thus present direct challenges to protein structure and function. By carefully optimizing the drying technique, for example, by using the lowest possible inlet temperature in the spray dryer, thereby giving a product with sufficiently low moisture content, the amount of heat applied to the product can be reduced. quantity can be minimized. Alternative drying methods such as freeze-drying or vacuum oven drying, which may introduce less heat to the protein and better preserve the native structure of the protein and ultimately the functionality of the protein in food systems. can be selected and optimized.

A protein concentrate or protein isolate slurry produced through precipitation or by membrane filtration can be spray dried to obtain a dry powder of protein concentrate or protein isolate. Spray drying involves passing the slurry through the spray dryer with the key operating process parameters (total solids content, feed flow, micronization air pressure, inlet and outlet temperature) optimized to achieve the desired moisture content of the powder. This can be done by passing

Example 4 Defatting of Protein Concentrates and Flours and Their Use for Protein Isolates Introduction:
The purpose of this example was to determine the most promising food-acceptable solvents in defatting protein flours and concentrates. Three common food grade organic solvents are tested: ethanol, isopropanol and hexane. The final product is measured for protein and lipid content to determine the degree of defatting success and the final protein concentration obtained.

Method:
Four grams of sample protein concentrate (PC) and protein-rich euglena flour (PF) were mixed in 40 mL of solvent (hexane, isopropanol, ethanol) on a stir plate for 24 hours at room temperature. Samples were then centrifuged (3500 rpm, 5 minutes). The supernatant was decanted into a new 50 mL tube. The pellet was resuspended in 40 mL of each solvent and vortexed for 20 seconds to mix. Samples were centrifuged again (3500 rpm, 5 minutes). The supernatant was retained in another 50 mL tube. Pellets and supernatant tubes were evaporated using a GeneVac EZ-2 evaporator. Samples of the starting material and defatted pellets were sent for total nitrogen analysis at the Water Quality Center at the University of Trent. Total lipids were extracted for delipidated and starting samples using an internal lipid extraction method for GC analysis that relies on chloroform and methanol to form a single-phase solvent system to extract and dissolve lipids. extraction was performed.

Results/Discussion:
The first major result was the observation that the crude fat analysis performed by SGS yielded significantly lower values (10-14% lower) than the total fat extraction method used internally. The major difference between these two extractions is that most of the euglenolipids in these samples are not soluble in ether (crude fat) and the 2 chloroform:2 methanol:1.8 suggesting that it was soluble in the water extraction mixture. The most likely reason in this instance is the presence of highly polar lipids such as phospholipids that have limited solubility in relatively non-polar ethers.

The protein concentrate defatted more readily than the flour, which went from 32.2% to about 20% lipid content after defatting in any of the solvents tested, and 48.4% total protein after defatting in any of the solvents tested. It became clear that it becomes about 20% from lipid. These results show that the concentrate had 75% of its lipids removed, whereas the flour had only about 50% of its lipids removed. These results are logical in the sense that the protein concentrate is homogenized and therefore the solvent dissolves the lipid components more readily than the semi-intact cells present in the flour. These results are supported by both Tables 11 and 12.

(Table 11) Lipid content before and after defatting

(Table 12) Lipid mass balance

The results in Tables 13 and 14 show that in this delipidation experiment, large amounts of protein were extracted into the various organic solvents tested, along with lipids. Hence, the lipid extract itself is unlikely to be purely lipid, possibly due to the large amount of lipoprotein complexes present in the sample. Further studies should examine the protein content of the evaporated lipid concentrates. Protein solubility was found to be highest in ethanol, both in protein concentrate and flour. For example, ethanol extraction lost 87.4% of the N content (surrogate for protein in this experiment) from the protein-rich flour to the solvent. It was unexpected that the protein had such high solubility in these solvents, and centrifugation was probably insufficient to recapture insoluble protein components. Alternatively, Euglena lipids may have an abnormally high N content (eg, phosphatidylcholine), causing this bias in the mass balance. However, this has to be explored further as it may reveal very interesting functionalities of Euglena proteins that are highly soluble in organic solvents. An option for recapturing suspended insoluble proteins may be to utilize centrifugation and filtration rather than centrifugation alone, such as gravity or vacuum filtration. Future studies could investigate the use of higher temperatures during extraction, which could disrupt interactions between lipids and proteins, thereby preventing protein carry-over in lipid extracts. .

(Table 13) Protein content before and after defatting

(Table 14) Protein mass balance

From the study in Example 3, it was observed that there was a 75% reduction in lipids and a 17.5% loss of protein with isopropanol extraction. Looking at a starting biomass of 49.3% protein, 48.4 lipids, and 2.3% other (such as ash and carbohydrates), applying the same logic we find 21.97% lipids and 4.13% other Assume that there is a final protein percentage of 73.9% containing Assuming no protein loss in solvent through modification, the protein percentage could be as high as 77.4%.

Defatting was found to be useful for color control in both protein concentrates (Fig. 1) and protein flours (Fig. 2). These results suggest that the color (yellow/orange) of Euglena powder is due to fat-soluble components such as carotenoids. Solvent defatting can be utilized to increase the neutrality of the protein's color attribute when color is important to the product application.

Other solvents that can be used to remove lipids from protein samples include acetone, benzyl alcohol, 1,3-butylene glycol, carbon dioxide, castor oil, citrate esters of mono- and diglycerides, ethyl acetate, ethyl Alcohol (ethanol), ethyl alcohol denatured with methanol, glycerol (glycerin), glyceryl diacetate, glyceryl triacetate (triacetin), glyceryl tributyrate (tributyrin), hexane, isopropyl alcohol (isopropanol), methyl alcohol (methanol), Methyl ethyl ketone (2-butanone), methylene chloride (dichloromethane), mono- and diglycerides, citric acid monoglyceride, 1,2-propylene glycol (1,2-propanediol), propylene glycol mono- and diesters of fat-forming fatty acids, and citric acid Contains triethyl.

Conclusion:
Protein concentrates and flours were defatted using ethanol, isopropanol and hexane. Protein concentrate defatted more easily than protein flour. Nitrogen may have been carried over into the lipid extract and interfered with the results, suggesting protein loss or high nitrogen content in the euglenolipids. The defatted residue was much more neutral in color than the starting material. Next steps included investigating the protein content of lipid extracts, comparing crude lipid extracts (petroleum ether) with the extracts prepared in these experiments, and investigating the effect of temperature on lipid extraction. to do.

Conclusions drawn regarding Euglena protein isolates:
This study shows that 75% of the fat in protein concentrate samples can be removed. The study also shows that, on average, without any modification, there is a protein (nitrogen) loss of 17.5% to 36.2% in the lipid extraction solvent, which varies based on the solvent used. Of the three tested here, isopropanol had the lowest protein loss at 17.5%. Based on this, materials with higher starting protein percentages with defatting by solvent extraction, as observed in Example 3, would yield Euglena protein isolates with more than 80% protein. It can be inferred that See, for example, the approximate data from Example 3, reproduced here in Table 15.

Table 15 Approximate Data from Pre-Homogenization Alkaline Extraction of Case 3

The protein concentrate powder is 70.4% protein, 21.9% fat, a small amount of carbohydrates and some ash. As shown in the calculations below, with the following assumptions, if this biomass were defatted by isopropanol solvent extraction, the protein isolate would be expected to have an average protein content of 81.5%. be.

For these calculations, a 100 g sample of Euglena protein concentrate powder was used and approximate data are shown in Table 15.

With isopropanol, 75% lipid removal is expected, 21.9g (0.75) = 16.4g lipid removed, leaving 5.5g lipid. If isopropanol is used, it is expected to have an average protein loss of 17.5%. So out of the original 70.4g protein: 70.4g (0.175) = 12.32g protein lost, leaving 58.1g protein. This example also assumes that there is no carbohydrate or ash loss in solvent extraction, meaning that 0.5 g of carbohydrate and 7.2 g of ash are still present. So the new total mass is: 58.1 g protein + 5.5 g fat + 7.2 g ash + 0.5 g carbohydrate = 71.3 g total mass.

Based on these assumptions, it is assumed that there are 58.1 g of protein in a 71.3 g sample, ie the sample contains 81.5% protein. The new lipid content is then also 7.7%, the carbohydrate content is 0.7% and the new ash content is 10.1%. This summary is found below in Table 16, which highlights changes in protein and lipid content. Thus, in this example the protein isolate has a protein content of 81.5%. Theoretically, protein content could be as high as 84.2% if solvent methods were improved to minimize protein loss in solvent extractions such as vacuum filtration. If the Euglena protein concentrate of Example 3, Table 8 were delipidated using the same logic as isopropanol, it would have a protein content of 82.9% assuming protein loss in the solvent, or The improved method produces a protein isolate with a protein content of 85.4%.

Table 16. Table summarizing conversion of protein concentrate to protein isolate by isopropanol solvent extraction. Numbers are given as percent dry basis.

Example 5: Amino Acid Profiles, Digestibility and PDCAAS Scores of Protein Flours and Protein Concentrates corrected amino acid score) values were examined. Samples were sent to a third-party analytical laboratory to determine the percentage of amino acids, total protein, and PDCAAS counts present in the samples.

Amino acid profiles, PDCAAS (Protein Digestibility Corrected Amino Acid Scores) and digestibility are reported in Table 17 for protein concentrates and protein flours. Total protein varied between 33% and 48% for the protein concentrate samples (n=2) and between 18% and 38% for the protein rich flours (n=4). As expected, total protein was on average higher in the protein concentrate samples compared to the protein-rich flours.

For protein concentrates, PDCAAS scores were 0.96 and 0.73. A value of 1 represents a perfect reference protein, so 0.96 is a very high score and 0.73 is still a good indication of protein quality. PDCAAS values ranged from 0.93 to 1.21 for the protein-rich flours. A PDCAAS score greater than 1 indicates that these samples have higher levels of essential amino acids. Since the protein in the protein-enriched Euglena flour is close to or greater than 1, this indicates that the protein in the protein-enriched Euglena flour is of high quality and high digestibility. PDCAAS scores greater than 1 are rounded down to 1 as an acceptable score.

アスタリスクは9つの必須アミノ酸を表す。アミノ酸値は、パーセンテージとして与えられている。 Table 17. Amino Acid Profiles, Digestibility, and PDCAAS Scores for Protein Concentrates and Protein Flours

Asterisks represent the 9 essential amino acids. Amino acid values are given as percentages.

Example 6: Egg Replacer Containing Euglena Protein Powder, Beta Glucan Isolate, and Ready-to-Gel Powder There are two different egg replacement product strategies in the market, one aimed at bakery applications. do. In that application, the main ingredients are starches and gums (used as binders and texturizers) along with leavening agents to supplement the leavening function of egg whites in baking applications, along with some proteins. .

Other uses for egg replacers are powdered or liquid egg replacers that can be scrambled for use as scrambled eggs or omelets. For scrambled egg substitutes, the main ingredients include vegetable protein (to mimic the nutrition of real eggs) and different gums/hydrophilic proteins as binders and texture developers when cooking. Contains a mixture of colloids.

In this study, a combination of pea protein concentrate and euglena protein powder was used as the main protein source. Beta-glucan ready-to-gel (RTG) powder was used as the sole hydrocolloid source to act as a binder/texturant. Beta-glucan RTG powder is solubilized beta-glucan in 1M NaOH that is formed into a gel containing 3.75% citric acid and then lyophilized to form a powder. When the powder is reconstituted with water, it immediately forms a thickened solution or gel, depending on the concentration. The addition of the β-glucan isolate in combination with the Euglena protein powder gives the egg scramble the expected yellow color of scrambled eggs, i.e. no coloring agents are required for these applications. Furthermore, the addition of β-glucan isolate also showed a masking effect on the off-note of Euglena protein powder compared to the control mixture without β-glucan isolate. Euglena flour is yellow, which also increases the expected yellow color of scrambled eggs.

No flavoring agents or flavor-masking substances were used in this formulation, yet the panelists were able to perceive an umami-like taste due to the protein source (combination of Euglena flour and pea protein).

Methods and Materials:
The formulations used in this study for egg scrambled are listed in Table 18 and consisted of Euglena protein flour with approximately 30% protein in the flour, β-glucan isolate from Euglena, and Euglena as a hydrocolloid source. Contains ready-to-gel beta-glucan powder to work. The dry ingredients are mixed together, then water is added and mixed. Whisk the mixture for 1 minute. To test the scramble-like properties, a frying pan set to medium heat (approximately 170°C) and 1 tablespoon of warmed oil (i.e. vegetables, 1 tablespoon per 100 grams of liquid egg replacer) Pour the whipped mixture inside. The whipped mixture is fried for about 7 to about 9 minutes with scrambling every 1 to 2 minutes to achieve the consistency of scrambled eggs. The mixture was analyzed by an internal taste panelist.

Table 18. Egg (Scrambled) Substitute Formulations

Results and Discussion:
The Euglena-based liquid scrambled egg substitute had a yellow color similar to the control scrambled egg. In this case, no flavoring agents or other known flavor masking agents were added to this formulation. Thus, in this formulation, taste panelists were able to detect an umami-like taste due to the protein sources of Euglena protein flour and pea protein. Onion powder or salt, garlic powder or salt, and/or nutritional yeast powder can be flavoring agents added to improve the taste of the Euglena liquid egg replacer.

Example 7: Further Studies on Liquid Egg Replacers In this study, the effects of gellan gum as a hydrocolloid modifier/binder, onion powder as a flavoring agent, and euglena flour enriched for higher protein content were investigated. investigated.

material and method:
Euglena liquid egg replacers are shown in Table 19. For Euglena protein flours, 37% protein and 48% protein flours were investigated to determine the effect of higher protein containing flours. No β-glucan isolate was used as a masking/whitening agent in this formulation. Also, RTG β-glucan powder was not used as a hydrocolloid. Instead, hydrocolloid gellan gum was added. An onion powder flavoring agent is also added to improve the taste of the final product.

All dry ingredients, including gellan gum, were mixed to form a mixture, followed by the addition of water and mixing. The mixture was heated to 80° C. for 1 hour and up to 2 hours for the gellan gum to hydrate and gel. Pour the mixture into a frying pan set to medium heat (approximately 170°C) and add 1 tablespoon of warmed oil (i.e. vegetables, 1 tablespoon per 100 grams of liquid egg replacer). The mixture is fried for about 7 to about 9 minutes with scrambling every 1 to 2 minutes to obtain the consistency of scrambled eggs. The mixture was analyzed by an internal taste panelist. The experiment was repeated again to confirm the results.

(Table 19) Ingredient list of Euglena liquid egg replacer

Results and Discussion:
After 1 hour of heat treatment of the lower content Euglena protein powder egg prototype (37% protein content), the presence of gellan gum produced a thick gel-like consistency similar to liquid egg, and the consistency of scrambled eggs when cooked. Aids desired texture. Even after 1 hour and about 2 hours under the same heat treatment conditions, the prototype with the higher Euglena protein content (47%) did not achieve the expected gel-like thick consistency and the mixture was less than before heat treatment. It was about the same consistency or only slightly thicker, too thin and watery for cooking. This effect was observed in all replicates of the experiment.

Conclusion:
Without wishing to be bound by theory, the results observed herein utilize higher concentrations of protein in the flour, such that the interaction between protein and gellan gum is prevented reconciliation. This means that the protein-gum interactions prevented the formation of water-gum interactions, preventing the hydration of the gums in the matrix and the thickening/gelling effect of the gums.

Example 8: Ready-to-Drink Beverage In this study, using Euglena protein powder as a protein source, the goal was to formulate a nutritious beverage with a few clean ingredients.

Methods and Materials:
Developed a chocolate drink formulation and tested different percentages of Euglena protein powder (3% w/w, 5% w/w, 7% w/w, 9% w/w final product) as sole protein source and evaluated the sensory profile (aroma, taste, mouthfeel) of the beverage.

All dry ingredients (protein powder, cocoa powder) and liquid ingredients (maple syrup and water) are weighed and combined in a glass container with a tight-fitting lid (see Table 20). Swirl or stir the mixture with a stir bar for about 2 minutes until all dry ingredients are dispersed and no lumps are observed. The beverage is homogenized for 2 minutes using a handheld (stand) homogenizer (ie OMNI International GLH-01) at speed setting 6, corresponding to 250,000 rpm. The homogenized beverage is then heated in an 80° C. water bath for 12 minutes. This time-temperature combination was achieved after several trials to identify the appropriate combination for the glass container containing the beverage to reach 72°C for 15 seconds (milk pasteurization criteria). The beverage is then chilled for 3 hours and tested for sensory profile.

(Table 20) Ready-to-drink beverages

Results and Discussion:
The above beverage formulation containing the Euglena product has 5.8 grams of Euglena protein/serving, 1.7% Euglena oil per serving, and 20.8 β-glucans per serving. Serving size is 200mL or 250mL.

With respect to the taste profile, the Euglena protein powder at the above concentrations produces a slight marine taste discomfort, but from higher concentrations the discomfort is less pronounced. Euglena β-glucan isolate was used in the formulation for its immunopotentiating effect. Euglena β-glucan isolate RTG was used in the formulation instead of including gum commonly used in beverage products to impart viscosity, mouthfeel and stability.

Example 9: Powdered Beverage Powdered Beverage Overview:
In this study, using Euglena protein powder as a protein source, we aim to formulate a nutritious powdered beverage with clean ingredients.

Powdered Beverage Problems and Solutions Agglomeration:
Euglena protein beverages contain pea protein, euglena flour, pregelatinized starch, masking substances, flavors and sugars. This blend forms large clumps when added to cold water. Tables 21 and 22 show the pregelatinized starch and Ticaloid 620 (locust bean from TIC) added to the formulation to achieve powdered beverage level viscosity (currently on the market) without agglomerates. gums and xanthan gum mixtures).

Marine Flavor Masking:
Various natural flavor masking agents and flavors were tested throughout the development of the powdered beverage. Tables 21 and 22 show the perfumes and masking substances most suitable for removing any marine flavor.

Table 21: Contents of Mango Flavored Powdered Protein Drink

Table 22: Contents of Chocolate Flavored Powdered Protein Drink

Suggestion Consumer Method and Offering Method:
The consumer receives a premixed package with multiple servings and a single serving. The consumer then prepares according to Table 23.

Table 23: Instructions for preparing one serving

Suggestions on how to serve/future use:
Tables 21 and 22 were tested with oat milk and 2% milk and the result was a thicker, more indulgent beverage formula. Thus, the versatility of powdered beverages and the potential for other possible uses (smoothies, baking, etc.) during future research into powdered beverages is demonstrated. Other milk or dairy substitutes suitable for beverages and tested during consumer testing include, but are not limited to, soy milk, soy milk blends, almond milk, lactose-free milk, coconut milk, cashew milk, and the like. not.

Conclusions and future work:
Noblegen's current powdered beverage overcomes texture and flavor barriers. Current flavors and formulations can be real competitors to current market products. A serving provides 12.6 g to 14.5 g of protein per serving in an easy to prepare application. Going forward, efforts will be made to powdered beverages to reduce the amount of sugar and add additional flavor to the current two. Studies on liquid beverages are also considered.

Example 10: Euglena Based Protein Bar The purpose of this study was to include Euglena protein powder in the bar formulation to replace part of the protein source and to give the bar a nutritional profile. (see Table 24).

Table 24: Ingredient List for Euglena Protein Powder Bar Formulation

To make the Euglena bar, heat the oven to 350° F. and bake the almonds and oats for 10 minutes or until just golden, then cool. After cooling, coarsely grind the almonds in a food processor, combine the dry ingredients in a mixing bowl, stir to combine, and set aside. Process dates in food processor until dough ball consistency forms, about 1 minute. Combine the moist ingredients (agave syrup, peanut butter, coconut oil, dates) in a small simmering double-cooker and heat until the dates are easily incorporated into the moist, then remove from heat. Using a spatula, add the wet ingredients into the dry ingredients until well mixed. Transfer the mixture into a suitably sized bar mold lined with wax paper and, using a spatula, press the dough firmly into the mold until evenly distributed and flat. Wrap the bar in wax paper and place in the refrigerator to harden.

Results and Discussion:
This formulation was developed by first testing 6.25% Euglena flour and 6.25% β-glucan isolate. The percentage of each was increased to 8% and 8.5% respectively, followed by 10% and 7% respectively. Formulations were also tested in which the β-glucan isolate was omitted from the recipe and the Euglena flour was increased to 12.5% 15%, 20%, 25% and 30%. To test the ability of 5% and 25% Euglena flour to retain water within the bar, the β-glucan isolate was reintroduced.

Below are previous formulation tests: maple syrup, agave syrup, tapioca and Golden Strap molasses, agave and tapioca syrup, beta-glucan isolate, ready-to-gel (RTG)-wet gel, and flavors. Describe masking.

Maple syrup:
Maple syrup was the first binder/sweetener used in bars derived from the original recipe. Maple syrup worked well, providing sufficient moisture to the bar and giving it a nice sweet flavor, but was not an ideal binder. Also, the shelf life was short. Maple syrup was used in all Euglena flour formulations (6.25% to 30%, listed above).

Agave syrup:
Agave syrup was used because it has a higher viscosity than maple syrup and may have better binding properties. This was observed to be the case. Additionally, agave syrup has a lower glycemic index than maple syrup and provides increased nutritional value for sustained energy. This bar contained 15%, 20%, 25% and 30% Euglena flour.

Tapioca and Goldenstrap Molasses:
This bar contained 30% Euglena flour. Tapioca and goldenstrap molasses mixes were tested as binders and expected to increase moisture while acting as a binder. This did not occur when equal amounts of tapioca and goldenstrap molasses were used as the moisture content in the formulation was too low. The dry ingredients were poorly moisturised and the resulting texture was lumpy, difficult to handle and off-flavoured. This bar contained 30% Euglena powder.

Agave and tapioca syrup:
A 50:50 ratio of agave and tapioca syrup mixture was used. While blending wet and dry, the mixture was found to be substantially drier than using agave alone. This was likely due to the fact that the tapioca syrup is quite viscous and does not contain much water. The formulation was adjusted to a 60:40 agave:tapioca distribution before combining the bars. All ingredients were added to the food processor to evenly distribute the new ratios before making the bars. Due to having more of the chopped ingredients on the surface, the food processor blended bars were more crumbly compared to the original packed bars. This bar contained 30% Euglena powder.

Beta Glucan Isolate (BGI):
The first bars were made utilizing BGI and Euglena flour at 6.25% each. Increasing the percentage of BGI and trying all of the above listed percentages showed little change. The final bar was blended without BGI and there was no significant difference (ie mouthfeel, taste). The percentage of Euglena flour was increased to obtain higher protein content in the bar formulation. Most recently, BGI has been added to bar formulations to determine if it helps retain moisture in the bar longer and increase shelf life. This was done utilizing 5% BGI and 25% Euglena flour with the addition of agave syrup as a binder.

Ready to Gel (RTG) - Wet Gel:
A ready-to-gel (RTG) bar was first made using 3.29% RTG, 30% Euglena flour, and maple syrup as a binder. RTG was included in wet form to try to help with the moisture content of the bar. Ideally, the bar requires 60:40 wet to dry ingredients. RTG is believed to increase the overall moisture ratio of the wet:dry ingredients of the bar as well as adding moisture to increase shelf life. However, it was observed that the bars were drier in appearance and were more brittle and flake off when cut. The RTG wet gel did not retain water as expected from the appearance, but increased the water content and appeared to improve the mouthfeel of the bar.

Additional formulations include various naturally derived flavor masking agents to reduce undesirable Euglena flavor, brown rice syrup, barley malt syrup, malt extract and malt extract to optimize moisture content, consistency and flavor. Various binders such as oat extract blends, as well as preservatives to increase the shelf life of the bar, such as potassium sorbate, will be tested.

Example 11: Further Examples of Euglena-Based Protein Bars Summary:
A prototype bar was made to model Euglena for high protein sweetening applications. Bars are 10 grams or more per serving, 5 grams derived from Euglena, vegan, gluten-free, and contain less than or as much sugar as protein (uncommon in many protein-rich bars) , Euglena has/should have the highest inclusion percentage.

All ingredients in the bars were selected either for their nutritional profile, texture/mouthfeel characteristics, flavor and/or combinations of these characteristics.

Challenges and Obstacles:
Most of the research into the bars went into perfecting flavor and mouthfeel. The first few formulations had an unattractive Euglena flavor and powdery mouthfeel. Natural flavors and low sugar binders that reduce the flavor of Euglena when added to formulations. Together with these two additions, several changes in the percentage inclusions of the ingredients helped form the current bar formulations seen in Tables 25-30.

Current bar formula:
Coconut citrus bar (Figure 3):
The following are examples of the ingredients and processes involved in making a coconut citrus bar, giving an example of euglena protein flour in a sweetened protein bar application. Citrus is an ideal flavoring for Euglena-based bars as it naturally tones done to any Euglena discomfort.

Table 25: Contents of Coconut Citrus Bars

Table 26: Recipe Instructions for Coconut Citrus Bars

This bar application contains 16.8% Euglena protein powder, which adds 5.22g of protein and 2.2g of beta-glucan to the bar. The total protein content in the bar is 10.95g and the total sugar content is 10.56g.

Dark chocolate, almond, and cranberry protein bars (Figure 4)

Table 27: Contents of Dark Chocolate, Almond, and Cranberry Protein Bars

Below are examples of the ingredients and processes involved in making dark chocolate, almond, and cranberry protein bars. The bitterness from dark chocolate helps reduce the amount of bitterness that can come from the Euglena aftertaste. A small amount of natural lemon flavor was added to the formulation to help offset the Euglena flavor.

(Table 28) Dark Chocolate Almond Cranberry Bar Instructions

This bar application contains 15.90% Euglena protein powder, which adds 4.92g of protein and 2.40g of beta-glucan to the bar. The total protein content of the bar is 11.20g and the total sugar content is 10.96g.

Peanut Butter Chocolate Chip Cookie Dough Bars The following are examples of the ingredients and processes involved in making peanut butter chocolate chip bars using Euglena flour. This provides an example of a protein bar that is tastier, higher in protein and more clearly labeled.

Table 29: Peanut Butter Chocolate Chip Cookie Dough Bar Ingredients

Table 30: Peanut Butter Chocolate Chip Cookie Dough Bar Instructions

Future research:
To date, Euglena has been used primarily in the form of pressed bars, but as the bar category includes baked bars, granola bars, energy balls and crisp bars, Research will continue to expand.

Euglena has already been incorporated into baked goods and is very promising for the baked bar market. In the future, research will also be conducted to reduce the sugar content and increase the protein content of current bars and any compressed bars.

Example 12: Raw noodles Introduction:
Noodle and pasta research has focused on making protein-rich Euglena noodles. The types of noodles under development are basic pasta, gluten-free pasta, vegan pasta, soba (soba noodles), and 30% vegan euglena noodles (still in the research stage).

Current Formulations Tables 31-34 below are the current formulations for each fabric. Testing was done in a consumer-style kitchen equipped with a Kitchen-Aid stand mixer and pasta roller attachment. Each dough was rolled out to level 4 thickness and cut into linguine.
Testing was conducted by establishing control formulations for each of the following noodle varieties. Then, depending on the dough, the primary flour type(s) was stepwise reduced by 5% to 30% and replaced with protein-rich euglena flour until the ideal inclusion percentage was found.

basic egg pasta dough

Table 31. Ingredients in Basic Egg Pasta Dough

At 20% content, the dough was slightly more difficult to knead and roll out, but was still workable by hand. The use of commercially available equipment helps element this problem. The cooking time was 4 minutes, similar to the control, and the Euglena flavor was pronounced, but not objectionable. The cooked texture was richer than the control, but still palatable. Figure 5 shows stretched egg dough, and as is evident from the figure, the higher the level of Euglena, the drier and more delicate the dough.

gluten free pasta dough

Table 32: Gluten Free Pasta Dough Ingredients

A loading of 10% was the highest loading achievable with the gluten-free flour. There was no difference in the texture of the raw dough compared to the control and it was also successfully cooked within 5 minutes of the same time as the control. The cooked dough had a very similar texture to the control with a slight Euglena aftertaste. As is evident from Figure 6, the gluten-free dough was denser in nature, with a maximum workable Euglena content of 20%.

egg-free pasta dough

Table 33. Ingredients in Egg-Free Pasta Dough

A content of 20% was ideal for egg-free pasta dough. Similar to the basic egg pasta dough (Table 31), the eggless pasta dough became progressively more difficult to roll out with higher Euglena content, but with commercial pasta equipment this is less of a problem. deaf. Compared to the control, this formulation took an extra minute to cook and had a low to moderate Euglena flavor. The cooked texture was denser than the control, but only slightly, and this could be corrected with a slightly longer cooking time. As Figure 7 shows, the inclusion of 20% Euglena in the egg-free formulation is dense compared to the control, but the edges are only torn and still stretched.

Soba/soba noodles

(Table 34) Ingredients in soba/soba noodles

Soba noodles are inherently denser than all-purpose flour-based noodles. For this reason, the maximum Euglena content that can be handled by hand is 10%. Anything over 10% will tear when fed through the pasta rollers. Cooking time did not differ from controls. Overall flavor and texture were similar to the control. The natural flavor of buckwheat helped remove many of the Euglena flavor characteristics.

Future research:
Going forward, the paste/noodle focus should be on 30% Euglena noodles while still maintaining 20g of protein. Natural masking substances can be used to reduce the flavor of Euglena. A key finding from the testing process was that Euglena flour produced a denser dough. Additional equipment may be required to work with the 30% Euglena formula and other texture optimized ingredients. Table 34 (soba/euglena noodles) was dared to test with ginger, garlic and miso. Although there was no Euglena aftertaste, the noodles produced a unique flavor, proving that Euglena has great potential in the noodle market.

With further testing and equipment, Euglena noodles can be successfully formed into a variety of different shapes (penne, rotini, spaghetti) or used in stuffed pasta dishes (ravioli, cannelloni).

There is also the possibility of using Euglena in various types of noodles such as udon, ramen, vermicelli, and yakisoba.

Example 13: MAC and Cheese Euglena macaroni and cheese prototypes are currently in development. The aim is to create a high protein, vegan and gluten free product. This is packaged in a box containing pre-measured portions of dried noodles and packets of powdered sauce. The consumer is responsible for cooking the pasta and hydrating the sauce with either water or milk of the consumer's choice (dairy or non-dairy). The protein goal is 12-15 grams of protein per serving, half of which should be from Euglena.

Task:
Taste and texture are two major issues being addressed during research on this prototype.

taste:
The natural flavor of Euglena flour has a savory note that complements the Euglena and serves to develop the "cheesy" flavor that we seek to obtain. will help us to reduce the unpleasant taste.

Texture:
Stabilizers and textures can be used to help obtain the ideal texture for the finished sauce.

Table 35 is an example of the work being done on the classic "cheddar cheese macaroni and cheese".

(Table 35) Current mix of macaroni and cheese

Example 14: Example of Euglena Extrusion Introduction:
Extruded products are a part of high-volume food processing used to make a variety of different foods such as pasta, cereals, breads, textured vegetable proteins (TVPs), and ready-to-eat snacks. form. Depending on the process used, the product can be puffed like an instant snack and/or textured like a TVP. In this example, Euglena protein flour was extruded with different ratios of pea protein, rice flour, and with or without a masking substance. This makes Euglena flour stand out as a possible ingredient replacement or as a functional ingredient for extrusion.

material and method:
Several different formulations using Euglena flour as an ingredient were tested, varying the content level of Euglena flour, pea protein and the use of masking substances (Table 36). Rice flour is added to help bind the mixture together while the mixed tocopherols are added to preserve freshness. Two different masking substances were also tested in the formulation to help create a more neutral flavor profile for the final product.

Using standard methods for dry extrusion, the product was extruded with a residence time of 0.5 minutes, but can be extruded up to 3 minutes. The final product has an expansion ratio of 0.3g/cc to 0.4g/cc.

(Table 36) Extrusion product formulations tested. In different versions, the pea protein, euglena protein and masking substance contents were varied.

Results and Discussion:
The extruded product yielded small crispy puffs that were crispy on the outside and firm on the inside. Similar results were seen between different ratios with or without masking material (Figures 8 and 9). This demonstrated good extrusion and puffing of the mixture with the Euglena protein powder.

The effectiveness of masking substances is primarily used for product quality. Version 6, with the highest content of masking substances, removed the marina flavor of Euglena. Masking materials have been shown to reduce discomfort over storage time, presumably by neutralizing volatile compounds in the mixture.

Conclusion:
This test produced small round puffs. Future formulations may vary the content of Euglena flour as protein up to 100%, ie up to 75%-80% in the formulation. Euglena protein concentrates and/or Euglena protein isolates can be used to replace the pea protein and/or to replace the Euglena flour in the formulation. It is possible to obtain all Euglena protein-based formulations, including mixtures of Euglena protein-rich flours, concentrates and isolates.

If the formulation is mixed with another protein, other protein isolates than pea can be used. Examples include soybeans, corn, wheat, rice, beans, seeds, nuts i.e. almond protein, peanut protein, seitan, lentils, chickpeas, flaxseed, wild rice, quorum, chia seeds, quinoa, oats. Barley, fava beans, buckwheat, bulgur, millet, microalgae, yellow peas, mung beans, hemp, sunflower protein, and legumes.

In addition, tapioca starch, rice starch, pea starch, corn starch, wheat starch, barley starch, sorghum starch, potato starch, sweet potato starch, turmeric starch, ginger starch, Dioscorea starch, water chestnut starch, arrowroot starch, oat starch, banana starch Other starches besides rice flour can also be used, such as lentil starch, yellow pea starch, chickpea starch, mung bean starch and amaranth starch.

The extruded puffs are often added as cereals, in cereal bars, granola bars, as a salad topping as an excellent source of protein in salads, and incorporated into baked goods. A suitable application for extruded puffs is puffed snacks, which can be coated with powdered flavorings to be eaten as ready-made food snacks.

When wet extrusion was used, wet extrusion also imparted texture to the product, including pasta, textured Euglena protein, plant-based muscle analogues, and mashed granules. The product list can be extended to other things such as pulled meat substitutes. In wet extrusion, instead of puffing the product by expansion, the goal is to texture the mixture through different standard manufacturing methods. One of the highlights is the use of crumbles of various sizes, or ingredients such as pulled pork or chicken that are 1/4-inch to 1-inch in size, to produce euglena protein-based TVP products. can be It can also be used to produce Euglena-based soy curls. The goal of meat substitutes is to create substitutes for all beef, pork, poultry and seafood based products. For seafood, to produce several different seafood applications such as crab/lobster, shrimp, clam, scallop, calamari, smoked fish, dried seaweed snacks and meat substitutes , can be combined with seaweed.

In conclusion, the version is successful in making a puffed extruded product and with further optimization, euglena-rich flour or euglena protein enrichment is possible. The masking substance helped stabilize the taste of the product over time.

Example 15: Content of Euglena Protein Flour, Concentrate, and Isolate in Various Foods (Table 37)

(Table 37) Food table. The points represent the form most likely to be used in the type of food, but are not limited to said forms, as is evident from the percentage values.

Table 38: Further Examples of Future Studies Using Euglena Protein Powders, Concentrates, and Isolates

Tables 37 and 38 highlight various functional uses of protein powders, protein concentrates and protein isolates from Euglena. Various methods of quality control and techniques such as those listed in Tables 37 and 38 are described below.

Emulsifying Activity To 5 mL of a 5% suspension of Euglena powder was added 5 mL of oil such as canola and the mixture was homogenized for 3 minutes. The emulsion was centrifuged at 500 rpm for 5 minutes. Emulsion layer height was measured using a scale on the centrifuge tube.

Emulsion stability:
Same procedure as for emulsifying activity, but samples were heated to 80° C. for 30 minutes in a water bath prior to centrifugation. The samples were then held under cold running water for 15 minutes.

Foaming capacity:
20 mL of 5% suspension was bubbled at 1600 rpm for 5 minutes. The mixture was poured into a 100 mL graduated cylinder and the foam volume was recorded.

Foam stability:
20 mL of 5% suspension was bubbled at 1600 rpm for 5 minutes. The mixture was poured into a 100 mL graduated cylinder and the foam volume was recorded after different time intervals ranging from 30 seconds to 60 minutes.

Apparent Viscosity:
Triplicate Euglena powder suspensions (45.0 +/- 0.05 g/255 ml distilled water) were made in 500 ml Pyrex beakers and mixed with a Servodyne mixer head at 800 rpm for 20 seconds using a Servodyne mix controller followed by hydrated for 5 minutes. Each mixture was poured into a 400 ml tall beaker and the viscosity was determined using a Brookfield Model LV digital viscometer equipped with a #1 or #2 disc spindle at 23C and 60 rpm. Viscosity readings were taken after the spindle had rotated for exactly 10 seconds.

PDI analysis (Ba10-65, AOCS1990):
Disperse duplicates of 20g of each sample in 300ml of distilled water at 25°C and 1°C. The dispersion was blended at 8,500 rpm for 10 minutes, poured into a 600 ml beaker and allowed to settle for 5 minutes. The upper liquid layer was decanted into a 50 ml glass centrifuge tube and centrifuged at 2900 rpm for 10 minutes. A LECO combustion analyzer was used to determine protein content in 250 mg of supernatant (% water-dispersible protein) and 250 mg of original Euglena flour (% total protein). The LECO device releases nitrogen from a sample by combustion in pure oxygen at high temperature. Liberated nitrogen is measured by a thermal conductivity detector and converted to percent protein by multiplying by a factor of 6.25. % PDI was calculated as follows.

Absorption of water:
10 mL of water was added to 0.5 g of protein in a 13 mL Sarstedt graduated plastic test tube. The mixture was sonicated for 30 seconds at a power setting of 5 to disperse the sample. The mixture was kept at 24°C for 30 minutes and then centrifuged at 2000 rpm for 25 minutes. The volume of free water was measured and the water retained was calculated and reported as ml of water absorbed per gram of powder (+/- 0.1 ml).

Fat absorption:
A 3 ml aliquot of peanut oil was added to 0.5 g of protein in a 13 ml graduated plastic test tube. The contents were sonicated for 1 minute at a power setting of 5 to disperse the sample. After 30 minutes at 24°C, the tubes were centrifuged at 2000 rpm for 25 minutes. The volume of oil released was measured and expressed as the oil retained in the flour pellets as ml absorbed per gram of flour (+/- 0.1 ml).

Gel strength:
The "torsion test" is a common test used to evaluate gel strength. An appropriately sized and shaped gel is twisted in the rheometer until the gel breaks or tears. The amount of force that split the cross-section is then calculated and can be measured against other sensory outcomes.

Gel strength is affected by temperature, pH and the amount of protein derivative in the food. Gel strength of foods containing protein flours, protein concentrates and/or protein isolates can be measured by a tensiometer. Through compression and tension data, a number of physical properties can be measured, including tensile strength, a measurement of the force required to pull a gelatinous or "gelled" food product to the breaking point, TA. Gel strength can also be measured by texture analyzers such as XT Express or TA.XTPlus (Texture Technologies), FTC Texture Analyzer (Food Technology Corporation) and LFRA Texture Analyzer (Brookfield Engineering). The Texture Analyzer also tests crunchiness, gumminess, adhesion, chewiness and general texture of many smaller items, from animal crackers to zucchini. Texture analyzers measure the tensile strength (ie, in pounds per ^inch2 or psi) and compressive strength (ie, psi or MPa) of materials. The principle of texture measurement systems is to physically deform a test sample in a controlled manner and measure its response. Force response properties are the result of mechanical properties of the sample that correlate to specific sensory textural attributes. The Texture Analyzer applies this principle by performing this procedure automatically and visually displaying the results on a digital numeric display or screen.

solubility:
Many solubility tests consist of suspending and stirring a known amount of protein in a buffered solution, followed by centrifugation to remove insoluble components, followed by protein analysis (colorimetric or Kjeldahl) of the supernatant. Based on

Methods for testing water retention capacity:
centrifuge:
A rapidly rotating device applies centrifugal force to the components for force separation. Fluids of different densities are thus separated, just as liquids are separated from solids.

Press method:
The water holding capacity of a food product is calculated based on the weight of the substance after it has been pressed.

Near-infrared spectroscopy non-destructively analyzes bulk high-moisture samples.

SERS (Surface Enhanced Raman Spectroscopy) will be utilized to detect the presence of harmful or toxic compounds in food.

A titrator will be used to test acidity and salt in foods.

Viscometers are used to test the viscosity of developed food products and effectively convey results related to mouthfeel, how the product reacts to temperature changes and the spread ability of the product.

The Bostwick Consistometer provides results on food consistency.

The disclosure of each patent, patent application, publication and accession number cited herein is hereby incorporated by reference in its entirety.

Preferences and preferences for any given aspect, feature, embodiment or parameter of the invention are to be combined with all preferences and preferences for all other aspects, features, embodiments and parameters of the invention unless the context dictates otherwise. should be considered to be disclosed as

Although the present disclosure has been disclosed with reference to various aspects, it will be apparent that other aspects and variations thereof can be devised by those skilled in the art without departing from the true spirit and scope of the disclosure. It is intended that the appended claims be interpreted to include all such aspects and equivalent variations.

Claims (50)

A composition comprising from about 5% to about 100% Euglena biomass. 2. The composition of claim 1, wherein said Euglena biomass is from about 10% to about 75% protein by dry weight. 2. The composition of claim 1, wherein said Euglena biomass is derived from heterotrophically cultured Euglena. 2. The composition of claim 1, wherein said Euglena biomass is predominantly intact Euglena cells. 6. The composition of claim 5, wherein the Euglena biomass is microalgal flour. 2. The composition of claim 1, wherein a protein concentrate is isolated from said Euglena biomass. 7. The composition of claim 6, wherein a protein isolate is isolated from said Euglena biomass or from said protein concentrate. 2. The composition of claim 1, wherein oil is extracted from said Euglena biomass. 2. The composition of claim 1, wherein said Euglena biomass is added to food. A food product comprising from about 0.1% to about 100% Euglena biomass and an edible ingredient. 11. The food product of claim 10, wherein said Euglena biomass is selected from the group consisting of microalgal flour, protein concentrate, and protein isolate. 12. The food product of claim 11, wherein said protein concentrate or said protein isolate is at least 40% protein. Sauces, teas, candies, cookies, cereals, breads, fruit mixes, fruit salads, salads, snack bars, protein bars, fruit leathers, yogurts, health bars, granola, smoothies, soups, juices, cakes, pies, shakes, ice cream 11. The food product of claim 10, selected from the group consisting of: , protein drinks, nutritional drinks, animal analogues, and health drinks. 11. The food product of claim 10, which is non-dairy selected from the group consisting of cheese, yogurt, milk, casein substitutes, whipped cream, protein drinks, non-dairy creamers, and combinations thereof. 11. The food product of claim 10, which is an animal analogue. The animal analogue is a meat analogue, a sausage analogue, a pepperoni/cured meat analogue, a chicken analogue, a turkey analogue, a pork analogue, a bacon analogue, a beef analogue, a tofu substitute, a ground beef analogue. , jerky analogues, egg analogues, egg replacers, hard-boiled egg replacers, powdered egg replacers, liquid egg replacers, frozen egg replacers, salad dressings, mayonnaise, and combinations thereof 16. The food product of claim 15. 11. The food product of claim 10, which is an extruded product selected from the group consisting of protein crisps, crackers, bars, pretzels, seaweed-like snacks, cereals, pastas, crisps, puffs, oatmeal, cookies, and combinations thereof. 11. The food product according to claim 10, which is a nutritional drink. 11. The food product of claim 10, which is a protein bar. A food product according to any one of claims 10 to 19, which is in liquid form. A food product according to any one of claims 10 to 19, which is in dry or powdered form. culturing Euglena;
Concentrating Euglena into microalgal biomass;
washing the microalgal biomass;
adjusting the pH of the microalgal biomass;
A method for preparing a microalgal flour, comprising drying a microalgal biomass to produce a microalgal flour. 23. The method of claim 22, wherein said microalgal biomass consists of about 25% to about 99% intact cells. 23. The method of claim 22, wherein the microalgal flour is from about 25% to about 55% protein by dry weight. culturing Euglena;
bringing the culture to about 1% to about 30% solids to form biomass;
adjusting the biomass to a pH of about 3 to about 11;
homogenizing the biomass;
centrifuging the homogenate;
separating a homogenate into three or more layers, the layers being a pellet, an aqueous middle layer, and a top lipid-enriched layer, for preparing a protein concentrate. a method of
the aqueous intermediate layer contains a soluble protein;
Method. 26. The method of claim 25, wherein said pellets contain a separate layer of protein concentrate sludge that is physically separated from aqueous and insoluble components. 26. The method of claim 25, wherein the pH of said aqueous intermediate layer is adjusted to about 3.5 to about 5.5. 28. The method of claim 27, wherein said aqueous intermediate layer is incubated at 22[deg.]C for at least 1 hour. 29. The method of claim 28, wherein said aqueous middle layer is further centrifuged to produce a protein concentrate sludge. 30. The method of claim 29, wherein said protein concentrate sludge is diluted with an equal weight of water to make a protein concentrate slurry. 31. The method of any one of claims 29-30, wherein the protein concentrate sludge or protein concentrate slurry is adjusted to a pH of about 5.5 to about 8.5. 32. The method of any one of claims 30-31, wherein said protein concentrate slurry is spray dried to produce said protein concentrate. 26. The method of claim 25, wherein said protein concentrate has a protein concentration that is at least 40% protein. culturing Euglena;
bringing the culture to about 1% to about 30% solids to form biomass;
adjusting the biomass to a pH of about 6 to about 11;
homogenizing the biomass;
centrifuging the homogenate;
separating the homogenate into three or more layers, the layers being a pellet, an aqueous middle layer, and a top lipid-enriched layer; a method for
the aqueous intermediate layer contains a soluble protein;
Method. 35. The method of claim 34, wherein said aqueous intermediate layer is adjusted to a pH of about 3.5 to about 5.5. 36. The method of claim 35, wherein said aqueous intermediate layer is incubated at 22[deg.]C for at least 1 hour. 37. The method of claim 36, wherein said aqueous middle layer is further centrifuged to produce a protein concentrate sludge. 38. The method of claim 37, wherein said protein concentrate sludge is diluted with an equal weight of water to make a protein concentrate slurry. 39. The protein concentrate slurry of claim 38, wherein said protein concentrate slurry is (a) centrifuged and (b) resuspended in water, and (a) and (b) are optionally repeated one or more times. the method of. 40. The method of any one of claims 37-39, wherein the protein concentrate sludge or protein concentrate slurry is adjusted to a pH of about 5.5 to about 8.5. 41. The method of any one of claims 38-40, wherein said protein concentrate slurry is spray dried, thereby producing said protein isolate. 42. The method of any one of claims 25-41, wherein said protein concentrate is defatted using an organic solvent, thereby increasing said protein content to greater than 80%. The organic solvent includes acetone, benzyl alcohol, 1,3-butylene glycol, carbon dioxide, castor oil, citrate esters of mono- and diglycerides, ethyl acetate, ethyl alcohol (ethanol), methanol-denatured ethyl alcohol, glycerol ( glyceryl), glyceryl diacetate, glyceryl triacetate (triacetin), glyceryl tributyrate (tributyrin), hexane, isopropyl alcohol (isopropanol), methyl alcohol (methanol), methyl ethyl ketone (2-butanone), methylene chloride (dichloromethane), monoglycerides and selected from the group consisting of diglycerides, citric acid monoglycerides, 1,2-propylene glycol (1,2-propanediol), propylene glycol mono- and diesters of lipogenic fatty acids, triethyl citrate, and combinations thereof. 42 described method. 42. The method of claim 41, wherein said protein isolate has a protein concentration that is at least 80% protein. 45. The method of any one of claims 34-44, wherein said pellet is greater than 95% beta-glucan. The Euglena is Euglena gracilis, Euglena sanguinea, Euglena deses, Euglena mutabilis, Euglena acus, Euglena viridis ), Euglena anabaena, Euglena geniculata, Euglena oxyuris, Euglena proxima, Euglena tripteris, Euglena chlamydophora, Euglena splendens, Euglena texta, Euglena intermedia, Euglena polymorpha, Euglena ehrenbergii, Euglena adhaerens, Euglena Euglena clara, Euglena elongata, Euglena elastica, Euglena oblonga, Euglena pisciformis, Euglena cantabrica, Euglena granulata Euglena granulata, Euglena obtusa, Euglena limnophila, Euglena hemichromata, Euglena variabilis, Euglena caudata, Euglena minima minima), Euglena communis, Euglena magni Euglena magnifica, Euglena terricola, Euglena velata, Euglena repulsans, Euglena clavata, Euglena lata, Euglena tuberculata ( Euglena tuberculata, Euglena contabrica, Euglena ascusformis, Euglena ostendensis or a combination thereof. A composition according to any one of claims 10-21, or a method according to any one of claims 22-45. 11. The composition according to claim 9 or the food product according to claim 10, wherein the Euglena biomass is dried Euglena biomass. 11. The composition according to claim 9 or the food product according to claim 10, wherein the Euglena biomass is wet Euglena biomass. 11. The composition of claim 1 or the food product of claim 10, further comprising flavorants, masking agents and/or additional ingredients. 35. The method of claim 22, claim 25, or claim 34, further comprising applying flavorants, masking agents, and/or additional ingredients to said culturing Euglena and/or said biomass.

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